Her-2 binding antagonists

ABSTRACT

There is disclosed a pharmaceutical composition for treating solid tumors that overexpress HER-2, comprising an agent selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1 or SEQ ID NO:12, wherein the polypeptide binds to the extracellular domain ECD of HER-2 at an affinity of at least 10 8 , (b) an isolated and glycosylated polypeptide having from about 300 to 419 amino acids taken from the sequence of SEQ ID NO:2 or SEQ ID NO:13, wherein the C terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present, (c) a monoclonal antibody that binds to the ECD of HER-2, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone, and pharmaceutically acceptable carrier. Also disclosed are prognostic and diagnostic assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/204,102 filed 10 Dec. 2002 (allowed, issued Fee paid), which is theUnited States national stage pursuant of 35 U.S.C. §371 ofPCT/US01/05327 filed 16 Feb. 2001, which is a continuation-in-part ofU.S. patent application Ser. No. 09/506,079, filed 20 Jan. 1999, all ofwhich are incorporated by reference herein in their entirety.

FEDERALLY FUNDING ACKNOWLEDGEMENT

This work was supported by a grant from the Department of Defense (DOD)Breast Cancer Research Program. The United States Government has certainrights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention provides a HER-2 binding antagonist. Specifically,intron retention has generated a novel HER-2 antagonist polypeptide thatbinds to the HER-2 receptor.

BACKGROUND

The HER-2/neu (erbB-2) oncogene encodes a receptor-like tyrosine kinase(RTK) that has been extensively investigated because of its role inseveral human carcinomas (Hynes and Stern, Biochim. et Biophys. Acta1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994) andin mammalian development (Lee et al., Nature 378:394-398, 1995). Thesequence of the HER-2 protein was determined from a cDNA that was clonedby homology to the epidermal growth factor receptor (EGFR) mRNA fromplacenta (Coussens et al., Science 230:1132-1139, 1985) and from agastric carcinoma cell line (Yamamoto et al., Nature 319:230-234, 1986).The HER-2 mRNA was shown to be about 4.5 kb (Coussens et al., Science230:1132-1139, 1985; and Yamamoto et al., Nature 319:230-234, 1986) andencodes a transmembrane glycoprotein of 185 kDa in normal and malignanthuman tissues (p185HER-2) (Hynes and Stern, Biochim. et Biophys. Acta1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994). Thefunction of the HER-2 gene has been examined mainly by expressing thecDNA corresponding to the 4.5 kb transcript in transfected cells andfrom the structure and biochemical properties of the 185 kDa proteinproduct. P185HER-2 consists of a large extracellular domain, atransmembrane segment, and an intracellular domain with tyrosine kinaseactivity (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994;and Dougall et al., Oncogene 9:2109-2123, 1994). Overexpression ofp185HER-2 causes phenotypic transformation of cultured cells (DiFiore etal., Science 237:178-182, 1987; and Hudziak et al., Proc. Natl. Acad.Sci. USA 84:7159-7163, 1987) and has been associated with aggressiveclinical progression of breast and ovarian cancer (Slamon et al.,Science 235:177-182, 1987; and Slamon et al., Science 244:707-712,1989). p185HER-2 is highly homologous to the EGFR. However, a ligandthat directly binds with high affinity to p185HER-2 has not yet beenidentified. Moreover, the signaling activity of HER-2 may be mediatedthrough heterodimerization with other ligand-binding members of the EGFRfamily (Carraway and Cantley, Cell 78:5-8, 1994; Earp et al., BreastCancer Res. Treat. 35:115-132, 1995; and Qian et al., Oncogene10:211-219, 1995).

Divergent proteins, containing regions of the extracellular domains ofHER family RTKs, are generated through proteolytic processing of fulllength receptors (Lin and Clinton, Oncogene 6:639-643, 1991; Zabrecky etal., J. Biol. Chem. 266:1716-1720, 1991; Pupa et al., Oncogene8:2917-2923, 1993; Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996;and Vecchi and Carpenter, J. Cell Biol. 139:995-1003, 1997) and throughalternative RNA processing (Petch et al., Mol. Cell. Biol. 10:2973-2982,1990; Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993; and Lee andMaihle, Oncogene 16:3243-3252, 1998). The extracellular domain ofp185HER-2 is proteolytically shed from breast carcinoma cells in culture(Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990; Scott et al., Mol.Cell. Biol. 13:2247-2257, 1993; and Lee and Maihle, Oncogene16:3243-3252, 1998), and is found in the serum of some cancer patients(Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992) where it is may bea serum marker of metastatic breast cancer (Leitzel et al., J. Clin.Oncol. 10:1436-1443, 1992) and may allow escape of HER-2-rich tumorsfrom immunological control (Baselga et al., J. Clin. Oncol. 14:737-744,1966; and Brodowicz et al., Int. J. Cancer 73:875-879, 1997).

A truncated extracellular domain of HER-2 is also the product of a 2.3kb alternative transcript generated by use of a polyadenylation signalwithin an intron (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993).The alternative transcript was first identified in the gastric carcinomacell line, MKN7 (Yamamoto et al., Nature 319:230-234, 1986; and Scott etal., Mol. Cell. Biol. 13:2247-2257, 1993) and the truncated receptor waslocated within the perinuclear cytoplasm rather than secreted from thesetumor cells (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993).However, no particular therapeutic, diagnostic or research utility hasbeen ascribed to this truncated extracellular domain polypeptide. Atruncated extracellular domain of the EGFR, generated by alternativesplicing (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990) issecreted, exhibits ligand-binding, and dimerization properties (Basu etal., Mol. Cell. Biol. 9:671-677, 1989), and may have a dominant negativeeffect on receptor function (Basu et al., Mol. Cell. Biol. 9:671-677,1989; and Flickinger et al., Mol. Cell. Biol. 12:883-893, 1992).

Therefore, there is a need in the art to find molecules that bind tocellular HER-2 and particularly molecules that bind to different sitesthan humanized antibodies to HER-2 (e.g., HERCEPTIN®). Such moleculeswould be useful therapeutic agents for various cancers that overexpressHER-2.

SUMMARY OF THE INVENTION

The present invention provides an isolated polypeptide having from about50 to 79 amino acids taken from the sequence of SEQ ID NO. 1, whereinthe polypeptide binds to the extracellular domain ECD of HER-2 at anaffinity of at least 10⁸. Preferably, the isolated polypeptide is fromabout 69 to 79 amino acids in length. Preferably, the isolatedpolypeptide binds to a site on the ECD of HER-2 that is different fromthe site of binding of HERCEPTIN® (a marketed humanized monoclonalantibody that is used for the treatment of cancer and that binds to theECD or HER-2).

The present invention further provides an isolated DNA sequence thatcodes, on expression, for a polypeptide having from about 50 to 79 aminoacids taken from the sequence of SEQ ID NO. 1, wherein the polypeptidebinds to the extracellular domain ECD of HER-2 at an affinity of atleast 10⁸. Preferably, the isolated polypeptide is from about 69 to 79amino acids in length. Preferably, the isolated polypeptide binds to asite on the ECD of HER-2 that is different from the site of binding ofHERCEPTIN® (a marketed humanized monoclonal antibody that is used forthe treatment of cancer and that binds to the ECD or HER-2). The presentinvention further provides a transfected cell comprising an expressionvector having a DNA sequence that codes on expression for a polypeptidehaving from about 50 to 79 amino acids taken from the sequence of SEQ IDNO. 1, wherein the polypeptide binds to the extracellular domain ECD ofHER-2 at an affinity of at least 10⁸.

The present invention further provides an isolated and glycosylatedpolypeptide having from about 80 to 419 amino acids taken from thesequence of SEQ ID NO. 2, wherein the C terminal 79 amino acids arepresent, and wherein at least three N-linked glycosylation sites arepresent. Preferably, the isolated polypeptide is from about 350 to 419amino acids in length and four N-linked glycosylation sites are present.Preferably, the isolated polypeptide binds to a site on the ECD of HER-2that is different from the site of binding of HERCEPTIN® (a marketedhumanized monoclonal antibody that is used for the treatment of cancerand that binds to the ECD or HER-2).

The present invention further provides an isolated DNA sequence thatcodes on expression for a polypeptide having from about 80 to 419 aminoacids taken from the sequence of SEQ ID NO. 2, wherein the C-terminal 79amino acids are present, and wherein at least three N-linkedglycosylation sites are present. Preferably, the isolated polypeptide isfrom about 350 to 419 amino acids in length and four N-linkedglycosylation are present. The present invention further provides atransfected cell comprising an expression vector having a DNA sequencethat codes on expression for a polypeptide having from about 80 to 419amino acids taken from the sequence of SEQ ID NO. 2, wherein theC-terminal 79 amino acids are present, and wherein at least threeN-linked glycosylation sites are present.

The present invention provides a method for treating a solid tumorcharacterized by overexpression of HER-2, comprising administering anagent that binds to the extracellular domain (ECD) of HER-2, wherein theagent is selected from the group consisting of (a) an isolatedpolypeptide having from about 50 to 79 amino acids taken from thesequence of SEQ ID NO. 1, wherein the polypeptide binds to theextracellular domain ECD of HER-2 at an affinity of at least 10⁸, (b) anisolated and glycosylated polypeptide having from about 80 to 419 aminoacids taken from the sequence of SEQ ID NO. 2, wherein the C-terminal 79amino acids are present, and wherein at least three N-linkedglycosylation sites are present, (c) a monoclonal antibody that binds tothe ECD of HER-2, and (d) combinations thereof, with the proviso thatthe agent cannot be the monoclonal antibody alone. Preferably, the solidtumor that overexpresses HER-2 is selected from the group consisting ofbreast cancer, small cell lung carcinoma, ovarian cancer and coloncancer. Preferably, the agent is the isolated polypeptide having fromabout 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1. Mostpreferably, the agent is a combination of the isolated polypeptidehaving from about 50 to 79 amino acids taken from the sequence of SEQ IDNO. 1 and the monoclonal antibody that binds to the ECD of HER-2.

The present invention further provides a pharmaceutical composition fortreating tumors that overexpress HER-2, comprising an agent selectedfrom the group consisting of (a) an isolated polypeptide having fromabout 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1,wherein the polypeptide binds to the extracellular domain ECD of HER-2at an affinity of at least 10⁸, (b) an isolated and glycosylatedpolypeptide having from about 80 to 419 amino acids taken from thesequence of SEQ ID NO. 2, wherein the C terminal 79 amino acids arepresent, and wherein at least three N-linked glycosylation sites arepresent, (c) a monoclonal antibody that binds to the ECD of HER-2, and(d) combinations thereof, with the proviso that the agent cannot be themonoclonal antibody alone, and pharmaceutically acceptable carrier.Preferably, the agent is the isolated polypeptide having from about 50to 79 amino acids taken from the sequence of SEQ ID NO. 1. Mostpreferably, the agent is a combination of the isolated polypeptidehaving from about 50 to 79 amino acids taken from the sequence of SEQ IDNO. 1 and the monoclonal antibody that binds to the ECD of HER-2.

The present invention further provides a method for targeting atherapeutic agent to solid tumor tissue, wherein the solid tumor tissueis characterized by overexpression of HER-2, comprising attaching thetherapeutic agent to an isolated polypeptide having from about 50 to 79amino acids taken from the sequence of SEQ ID NO. 1, wherein thepolypeptide binds to the extracellular domain ECD of HER-2 at anaffinity of at least 10⁸. Preferably, the isolated polypeptide is fromabout 69 to 79 amino acids in length. Preferably, the isolatedpolypeptide binds to a site on the ECD of HER-2 that is different fromthe site of binding of HERCEPTIN® (a marketed humanized monoclonalantibody that is used for the treatment of cancer and that binds to theECD or HER-2).

The present invention further provides a method for determining theprognosis of tumor treatment in a patient for a tumor that overexpressesHER-2, comprising: (a) obtaining a bodily fluid sample from the patient,wherein the bodily fluid is selected from the group consisting of blood,serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilicalcord tissue, amniotic fluid, chorionic villi tissue, and combinationsthereof and (b) measuring the amount of p68HER-2 expressed using ananti-p68HER-2 antibody-based assay, wherein the assay is selected fromthe group consisting of ELISA, immunoprecipitation,immunohistocytochemistry, and Western analysis. Preferably, the methodfor determining the prognosis of tumor treatment further comprisesmeasuring the amount of p185HER-2 ECD in the bodily fluid, anddetermining a ratio between the amount of p68HER-2 and p185HER-2.

The present invention further provides an assay for cancer treatment,prognosis or diagnosis in a patient comprising: (a) obtaining a bodilyfluid sample from the patient, wherein the bodily fluid is selected fromthe group consisting of blood, serum, urine, lymph, saliva, tumortissue, placental tissue, umbilical cord tissue, amniotic fluid,chorionic villi tissue, and combinations thereof; (b) determiningwhether a particular ECDIIIa variant sequence is present in the bodilyfluid sample with a sequence identity assay; and (c) correlating thepresence of the ECDIIIa variant sequence to cancer treatment anddiagnosis using an historical database. Preferably, the sequenceidentity assay is selected from the group consisting of DNA sequencing,PCR assays, ELISA immunologic assays, immunoassays, hybridizationassays, and combinations thereof.

The present invention further provides an assay for cancer treatment,prognosis or diagnosis in a patient comprising: (a) obtaining a bodilyfluid sample from the patient, wherein the bodily fluid is selected fromthe group consisting of blood, serum, urine, lymph, saliva, tumortissue, placental tissue, umbilical cord tissue, amniotic fluid,chorionic villi tissue, and combinations thereof; (b) determiningwhether an amount of an p68HER-2 ECDIIIa variant is present in thebodily fluid sample using an anti-p68HER-2 antibody-based assay, whereinthe assay is selected from the group consisting of ELISA,immunoprecipitation, immunohistocytochemistry, and Western analysis; and(c) correlating the presence or amount of the p68HER-2 ECDIIIa variantto cancer treatment and diagnosis using an historical database.

The present invention further provides for the above-mentioned cancertreatment, prognostic or diagnostic assays, further comprising measuringthe amount of p185HER-2 ECD in the bodily fluid sample.

The present invention further provides for the above-mentioned cancertreatment, prognostic or diagnostic assays further comprising measuringthe amount of p185HER-2 ECD in the bodily fluid sample, and determininga ratio between the amount of p185HER-2 ECD and a particular p68HER-2ECDIIIa variant.

The present invention further provides for antibodies specific forECDIIIa variants of the sequence in SEQ ID NO:1 or SEQ ID NO:2, below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence and amino acid of the insert in theextracellular domain of HER-2. The HER-2 ECD coding sequence from exon1-9 (primers A and B) was amplified by PCR from a cDNA library fromSKOV-3 cells. A product of ˜1420 bp was found to be HER-2-specific bySouthern blot analysis. This product was subcloned and the nucleotidesequence was determined. In panel A, a nucleotide sequence (287 bp; SEQID NO:15) is shown for the 275 bp insert (within the open-ended boxes)plus the immediately adjacent 5′ and 3′ sequences (framed by theopen-ended boxes). The 275 bp insert sequence, using the numbering ofCoussens et al. (Science 230:1132-1139, 1985), is located betweennucleotide residues 1171 and 1172 and following amino acid residue 340in p185HER-2. SEQ ID NO:16 (276 bp) shows the 275 bp insert sequenceplus the immediately 5′ nucleotide (“G”). The consensus 5′ and 3′ splicesites at the arrows are shown in larger print. The inserted sequence isin-frame with 5′HER-2 exon sequence and is deduced to encode a 79 aminoacid extension (SEQ ID NO:14) following Arg 340 (R³⁴⁰). The novel 79acid sequence (SEQ ID NO:14) encoded by the insert is proline-rich (19%)and has a consensus asparagine linked glycosylation site, which isunderlined. A stop codon was found at nucleotides 236-238 within theinserted sequence. In panel B, the predicted product of the alternativetranscript is a truncated secreted protein which contains subdomains Iand II identical to p185 and is missing the transmembrane domain andcytoplasmic domain. If fully glycosylated, the expected size is 65-70kDa. This polypeptide product is referred to as p68HER-2. Thus, theproduct will be a truncated secreted protein which is missing thetransmembrane domain and cytoplasmic domain found in p185HER-2.

FIG. 2 shows the detection of alternative HER-2 transcripts containingthe ECDIIIa sequence by Northern blot analysis. PolyA+ mRNA (2.5 μg)from different human fetal tissues (Clontech) or isolated from HEK-293cells was resolved in a formalin agarose gel and transferred to aBRIGHTSTAR® membrane (Ambion) in 10×SSC. The membrane was hybridizedwith a ³²P-labeled antisense RNA probe complimentary to the ECDIIIsequence, stripped and reprobed with a ³²P-labeled cDNA probe specificfor the 5′ HER-2 exon sequence. The membranes were washed under highstringency conditions and analyzed by phosphorimaging (MolecularDynamics).

FIG. 3 shows a sequence-specific reactivity of anti-ECDIIIa with aprotein of ˜68 kDa in a human embryonic kidney cell line (HEK293). Cellextract protein (20 μg) and 20 μl of media conditioned by HEK-293 cellswere Western blotted and probed with anti-ECDIIIa diluted 1:10,000(lanes 1 and 2) or with anti-ECDIIa diluted 1:10,000 containing 50 μg/mlpurified His-tagged ECDIIIa peptide (lanes 3, 4).

FIG. 4 shows the expression of p185HER-2, relative to p68ECDIIIaexpression, is markedly elevated in carcinoma cell lines in which theHER-2 gene is amplified. Cell extracts (15 μg of protein) from humanembryonic kidney cell line (HEK293), nontumorigenic ovarian surfaceepithelial cell line (IOSEVAN), ovarian carcinoma cell line with HER-2gene amplification (SKOV-3), nontumorigenic breast epithelial cell line(HBL100), and breast carcinoma cell lines with HER-2 gene amplification(BT474 and SKBR-3), were resolved by SDS-PAGE in 7.5% acrylamide gelsand analyzed as a Western blot. The Western blot was probed with bothantibodies specific for p68HER-2 (anti-ECDIIIa) and for p185HER-2(anti-neu(C)).

FIG. 5 shows that p68ECDIIIa binds to p185HER-2. In panel A: Two mg ofSKBR-3 cells extracted in nondenaturing buffer were immunoprecipitatedwith 5 μl anti-neu(N) specific for the N-terminal sequence of p68HER-2and p185HER-2, or with 5 μl anti-neu(C) specific for the C-terminus ofp185HER-2 and then probed as a Western blot with both anti-ECDIIIaspecific for p68HER-2 and with anti-neu(C) specific for p185HER-2. Inpanel B: 100 μg of 17-3-1 cell extract were incubated in duplicate with50 μl packed volume of NiNTA agarose (Qiagen) coupled to 20 μg ofHis-tagged ECDIIIa or to 20 μg His-tagged CREB fragment in 200 μl ofwash buffer (20 mM Tris pH 8.0, 300 mM NaCl) at room temperature for 1hr with shaking. The resin was then washed 4 times with 500 μl of washbuffer and proteins were eluted by incubation with 50 μl SDS-samplebuffer at 100° C. for 2 min. Eluted proteins were analyzed by Westernblot analysis using antibodies against the C-terminus of p185HER-2,anti-neu(C). In panel C: Monolayers of ˜10⁵ 3T3 cells or HER-2transfected 17-3-1 cells in 12 well plates were washed twice with PBSand then incubated with 0.5 ml of serum-free media with 1% BSA and 39,75, 150, and 300 nM of purified recombinant His-tagged ECDIIIa for 2 hrsat 4° C. Cells were washed 1 time in PBS containing 1% BSA and twice inPBS and then were extracted in denaturing buffer. Equal aliquots (20 μgprotein) were analyzed by western blotting with antibodies specific forECDIIIa (anti-ECDIIIa) or, in the upper panel, with antibodies specificfor p185HER-2 (anti-neu(C)).

FIG. 6 shows that neither p68-rich conditioned media nor the ECDIIIapeptide stimulate tyrosine phosphorylation of p185HER-2. Monolayercultures of ˜10⁵ HER-2 transfected 17-3-1 cells were washed twice withPBS, incubated in serum-free media at 37° C. for 24 hrs, and thentreated for 10 minutes with 75 or 150 μM His-tagged ECDIIIa or with50×CM from HEK-293 cells that secrete high levels of p68 or 50×CM fromSKOV-3 cells that have no detectable p68HER-2. The treated cells wereextracted with denaturing buffer containing the phosphotyrosinephosphatase inhibitor vanadate (2 mM) and 20 μg/ml of cell extractprotein from each sample were analyzed by Western blot analysis withmonoclonal antibodies against phosphotyrosine (Sigma). The blot wasstripped by incubation at 55° C. for 30 min in 62.5 mM Tris pH 6.7, 2%SDS, and 100 mM 2-mercaptoethanol and then reprobed with anti-neu(C)specific for p185HER-2.

FIG. 7 shows that p68HER-2 inhibited anchorage independent growth oftumorigenic cells. SKOV-3 ovarian carcinoma cells and HER-2 transfected17-3-1 cells were suspended in media with 10% fetal bovine serumcontaining 0.3% agar (control conditions) to which was added 50×concentrated media conditioned by SKOV-3 cells (which contains nodetectable p68HER-2 (−p68 CM)), or 50× concentrated media conditioned byHEK-293 cells (which contains 20 nM p68HER-2 (+p68 CM)). Five times 10³cells were plated in triplicate for each experimental condition onto a0.5 ml layer of media containing 0.5% agarose in 12 well plates. Theresults shown are plotted as the mean and standard deviation of thenumber of colonies with more than 50 cells in triplicate wells countedat 21 days of incubation. Similar results were observed in threeseparate experiments.

FIG. 8 shows the nucleotide (SEQ ID NO:17) and deduced amino acidsequence (SEQ ID NO:18) of HER-2 Intron 8. Human genomic DNA wassubjected to PCR using primers that flank intron 8. PCR parameters were30 cycles of 94° C. for 1 min, 62° C. for 1 min, 72° C. for 30 s,followed by 1 cycle of 72° C. for 7 min. A 410 bp product was gelpurified and sequenced in the forward and reverse directions. Thesequence shown is the most common sequence found within intron 8 fromabout 15 different individuals. Positions of sequence variationresulting in amino acid substitutions as disclosed herein are marked byXs below the sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the initial discovery of analternative HER-2 mRNA of 4.8 kb with a 274 bp insert identified asintron 8. The retained intron is in-frame and encodes 79 amino acids(SEQ ID NO:1) followed by a stop codon at nucleotide 236. Thealternative mRNA predicts a truncated HER-2 protein that lacks thetransmembrane and intracellular domains and contains 419 amino acids(SEQ ID NO:2); 340 residues that are identical to the N-terminus ofp185HER-2 (SEQ ID NO:13) and 79 unique residues at the C-terminus (SEQID NO:1). Using specific antibodies against either the novel 79 aminoacid residue C-terminal sequence (SEQ ID NO:1) or the N-terminus ofp185HER-2, a 68 kDa protein product was identified (SEQ ID NO:2). This68 kDa protein is the product of an alternative HER-2 transcript, and isfound in cell extracts and in extracellular media from several celllines. Expression of the alternative transcript was highest in anontransfected human embryonic kidney cell line.

The results presented here show expression of alternative HER-2 mRNA,which contains an additional 274 nucleotides, probably intron 8.Consistent with this finding, an alternative transcript of ˜4.8 kb wasdetected in human fetal kidney tissue and in the human embryonic kidneycell line, HEK 293. Moreover, a transcript of 2.6 kb, which is the sizeexpected if the sequence is retained in the 2.3 kb truncated HER-2 mRNA(Yamamoto et al., Nature 319:230-234, 1986; and Scott et al., Mol. Cell.Biol. 13:2247-2257, 1993), was detected in human fetal liver tissue byNorthern blot analysis using a probe specific for the inserted sequenceor for the HER-2 ECD coding sequence (FIG. 2). The inserted sequenceintroduces a termination codon and predicts a novel 79 amino acidextension designated ECDIIIa at residue 340 of the p185HER-2 protein.The predicted protein therefore lacks the transmembrane andintracellular domains, but contains subdomains I and II of theextracellular domain of p185HER-2. As predicted, a secreted protein thatcontains N-terminal sequence of p185HER-2 and the C-terminal extensionprovided by the inclusion of the novel sequence was detected (FIGS. 3and 5). The ECDIIIa protein was found to be 68 kDa which is theapproximate size expected of the protein encoded by the alternativetranscript if the five N-linked glycosylation sites found in subdomainsI and II of p185HER-2 are glycosylated (Stern et al., Mol. Cell. Biol.6:1729-1740, 1986).

The data presented herein demonstrate that p68HER-2 specifically bindsto p185HER-2. The association with p185HER-2 may be conferred by thenovel proline rich ECDIIIa domain rather than the N-terminal subdomainsI and II of p68HER-2. While the HER-2 ECD, generated by in vitrodeletion mutagenesis, also contains subdomains I and II, it does notassociate with the extracellular domain of p185HER-2 unless engineeredto enhance their proximity (Tzahar et al., EMBO J. 16:4938-4950, 1997;O'Rourke et al., Proc. Natl. Acad. Sci. USA 94:3250-3255, 1997; andFitzpatrick et al., FEBS Letters 431:102-106, 1998). However, the uniqueECDIIIa peptide binds with high affinity (nM concentrations) top185HER-2 and to transfected 17-3-1 cells that overexpress p185HER-2(FIG. 5). Preferential binding of the ECDIIIa domain peptide to 17-3-1cells indicates that secreted p68HER-2 interacts with the extracellularregion of p185HER-2 at the cell surface. Therefore, p68HER-2 andfragments thereof appear to be a naturally occurring HER-2 bindingprotein, encoded by the HER-2 gene. In contrast to EGFR family ligands(Groenen et al., Growth Factors 11:235-257, 1994), p68HER-2 lacks an EGFhomology domain and contains the first 340 amino acids of the receptoritself, p185HER.

Previously described putative HER-2 ligands were found to associateindirectly with p185HER-2 only in a heterodimer with an EGFR familymember (Heldin and Ostman, Cytokine Growth Factor Rev. 7:33-40, 1996).Although it is possible that ECDIIIa binds indirectly to p185HER-2through a coreceptor, this seems unlikely since detergent solubilizedp185HER-2 was specifically and efficiently “pulled down” by immobilizedECDIIIa peptide (FIG. 5B).

For all naturally occurring or engineered ligands for mammalian EGFRfamily members, binding is tightly coupled to stimulation of receptordimerization and tyrosine phosphorylation (Hynes and Stern, Biochim. etBiophys. Acta 1198:165-184, 1994; Dougall et al., Oncogene 9:2109-2123,1994; and Groenen et al., Growth Factors 11:235-257, 1994). Althoughthey bind, neither p68HER-2 nor the ECDIIIa peptide was found toactivate p185HER-2. Activation was assessed in two different cell linesthat differ in the extent of p185HER-2 tyrosine phosphorylation,transfected 17-3-1 cells as well as SKOV-3 ovarian carcinoma cells.Furthermore in vitro self-phosphorylation activity, which is enhanced indimeric forms of p185HER-2 (Dougall et al., Oncogene 9:2109-2123, 1994;and Lin et al., J. Cell. Biochem. 49, 290-295, 1992), was not stimulatedby p68HER-2 or ECDIIIa. Similarly, the Argos protein, which is anextracellular inhibitor of the Drosophila EGF receptor and the onlyknown antagonist of class I RTKs, did not simulate tyrosinephosphorylation of the receptor (Schweitzer et al., Nature 376:699-702,1995). Likewise, Angiopoietin-2, a natural antagonist for the Tie 2 RTK,bound the endothelial receptor but failed to activate it (Maisonpierreet al., Science 277:55-60, 1997).

Without being bound by theory, since p68HER-2 occupies but does notactivate, it could block dimerization of p185HR-2. By analogy, HER-2ECD, when engineered to enhance its binding to RTKs, prevented theformation of productive dimers required for transphosphorylation andreceptor activation thereby having a dominant negative effect (O'Rourkeet al., Proc. Natl. Acad. Sci. USA 94:3250-3255, 1997). In contrast tothe HER-2 ECD, soluble p68HER-2 exhibited strong binding to p185HER-2,yet also contains subdomain I and II of the ECD. Since subdomain I maybe the low affinity, promiscuous ligand binding site required forrecruitment of p185HER-2 into heteromeric complexes (Tzahar et al., EMBOJ. 16:4938-4950, 1997), p68HER-2 could block this site and therebyobstruct recruitment of p185HER-2 into dimers. Alternatively, p68HER-2could compete with an uncharacterized ligand for binding to p185HER-2.The tissue-specific expression of p68HER-2 in human fetal liver andkidney may function to modulate the extent to which p185HER-2 isoccupied during development of these organs. Moreover, theoverexpression of p185HER-2, relative to p68HER-2 in tumor cells withHER-2 gene amplification (FIG. 3), could occur though a selectivepressure based on overcoming the effects of a binding protein such asp68HER-2. Therefore, p68HER-2 is the first example of a naturallyoccurring p185HER-2 binding protein that may prevent activation ofp185HER-2.

Pharmaceutical Composition

The present invention further provides a pharmaceutical composition fortreating solid tumors that overexpress HER-2, comprising an agentselected from the group consisting of (a) an isolated polypeptide havingfrom about 50 to 79 amino acids taken from the sequence of SEQ ID NO. 1,wherein the polypeptide binds to the extracellular domain ECD of HER-2at an affinity of at least 10⁸, (b) an isolated and glycosylatedpolypeptide having from about 300 to 419 amino acids taken from thesequence of SEQ ID NO. 2, wherein the C terminal 79 amino acids arepresent, and wherein at least three N-linked glycosylation sites arepresent, (c) a monoclonal antibody that binds to the ECD of HER-2, and(d) combinations thereof, with the proviso that the agent cannot be themonoclonal antibody alone, and pharmaceutically acceptable carrier.Preferably, the agent is the isolated polypeptide having from about 50to 79 amino acids taken from the sequence of SEQ ID NO. 1. Mostpreferably, the agent is a combination of the isolated polypeptidehaving from about 50 to 79 amino acids taken from the sequence of SEQ IDNO. 1 and the monoclonal antibody that binds to the ECD of HER-2.

The inventive pharmaceutical composition, comprising either or both ofthe inventive polypeptides and/or monoclonal antibody, can beadministered to a patient either by itself (complex or combination) orin pharmaceutical compositions where it is mixed with suitable carriersand excipients. Inventive polypeptide can be administered parenterally,such as by intravenous injection or infusion, intraperitoneal injection,subcutaneous injection, or intramuscular injection. Inventivepolypeptide can be administered orally or rectally through appropriateformulation with carriers and excipients to form tablets, pills,capsules, liquids, gels, syrups, slurries, suspensions and the like.Inventive polypeptide can be administered topically, such as by skinpatch, to achieve consistent systemic levels of active agent. Inventivepolypeptide is formulated into topical creams, skin or mucosal patch,liquids or gels suitable to topical application to skin or mucosalmembrane surfaces. Inventive polypeptide can be administered by inhalerto the respiratory tract for local or systemic treatment of cancerscharacterized by overexpressing HER-2.

The dosage of inventive polypeptide suitable for use with the presentinvention can be determined by those skilled in the art from thisdisclosure. Inventive polypeptide will contain an effective dosage(depending upon the route of administration and pharmacokinetics of theactive agent) of inventive polypeptide and suitable pharmaceuticalcarriers and excipients, which are suitable for the particular route ofadministration of the formulation (i.e., oral, parenteral, topical or byinhalation). The active inventive polypeptide is mixed into thepharmaceutical formulation by means of mixing, dissolving, granulating,dragee-making, emulsifying, encapsulating, entrapping or lyophilizingprocesses. The pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the inventive polypeptide in water-solubleform. Additionally, suspensions of the inventive polypeptide may beprepared as oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. The suspension may optionally contain stabilizersor agents to increase the solubility of the complex or combination toallow for more concentrated solutions.

Pharmaceutical formulations for oral administration can be obtained bycombining the active compound with solid excipients, such as sugars(e.g., lactose, sucrose, mannitol or sorbitol), cellulose preparations(e.g., starch, methyl cellulose, hydroxypropylmethyl cellulose, andsodium carboxymethyl cellulose), gelaten, gums, or polyvinylpyrrolidone.In addition, a desintegrating agent may be added, and a stabilizer maybe added.

Processes for Synthesizing p68 and 79 aa C Terminal Region

Polypeptide synthesis is done by a group of standard procedures forpolypeptide synthesis by sequential amino acids building through peptidesynthesis equipment, following manufacturer's instructions forsynthesizing peptides. Preferably, shorter polypeptides, of less than100 amino acids, are best suited for the method of synthesis throughsequential amino acid building of polypeptides. In addition,heterologous polypeptides can be expressed by transformed cells usingstandard recombinant DNA techniques to transform either prokaryotic oreukaryotic cells, provide appropriate growth media for their expression,and then purify the inventive polypeptide either from the media or fromintracellular contents depending upon the type of cell used and itsexpression characteristics.

Methods for Treating Cancer with p68, 79 aa C Terminal Region, andCombinations

The present invention provides a method for treating a solid tumorcharacterized by overexpression of HER-2, or HER-2 variants (see Example8) comprising administering an agent that binds to the extracellulardomain (ECD) of HER-2, wherein the agent is selected from the groupconsisting of (a) an isolated polypeptide having from about 50 to 79amino acids taken from the sequence of SEQ ID NO. 1, wherein thepolypeptide binds to the extracellular domain ECD of HER-2 at anaffinity of at least 10⁸, (b) an isolated and glycosylated polypeptidehaving from about 300 to 419 amino acids taken from the sequence of SEQID NO. 2, wherein the C terminal 79 amino acids are present, and whereinat least three N-linked glycosylation sites are present, (c) amonoclonal antibody that binds to the ECD of HER-2, and (d) combinationsthereof, with the proviso that the agent cannot be the monoclonalantibody alone. Preferably, the solid tumor that overexpresses HER-2 isselected from the group consisting of breast cancer, small cell lungcarcinoma, ovarian cancer, prostate cancer, gastric carcinoma, cervicalcancer, esophageal carcinoma, and colon cancer. Preferably, the agent isthe isolated polypeptide having from about 50 to 79 amino acids takenfrom the sequence of SEQ ID NO. 1. Most preferably, the agent is acombination of the isolated polypeptide having from about 50 to 79 aminoacids taken from the sequence of SEQ ID NO. 1 and the monoclonalantibody that binds to the ECD of HER-2.

The p68HER-2 polypeptide described herein was found to bind to HER-2 andprevent signal transduction through the kinase domain. Without beingbound by theory, the unique ECDIIIa domain mediates specific binding top185HER-2 and the resulting interaction with p68ECDIIIa preventsp185HER-2 dimerization and subsequent signal transduction. Therefore,p68HER-2 functions as a HER-2 antagonist to prevent signal transductionby preventing dimerization as a necessary prerequisite for signaltransduction. Thus, the mechanism of p68HER-2 as a HER-2 antagonist isdifferent from the mechanism of binding agents, such as the 79 aminoacid polypeptide described herein or a monoclonal antibody that binds tothe EDC of HER-2. The inventive method provides that p68HER-2 inhibitstumor cell growth in tumors that overexpress HER-2 by providing aselective pressure for such tumor cells. Similarly, the HER-2antagonists that are binding agents also inhibit tumor cell growth intumors that overexpress HER-2 by providing selective pressure to suchcells to prevent ligand binding to the ECD of HER-2 and prevent signaltransduction even before potential dimerization.

Use of 79 aa C Terminal Region as a Targeting Molecule

The present invention further provides a method for targeting atherapeutic agent to solid tumor tissue, wherein the solid tumor tissueis characterized by overexpression of HER-2, comprising attaching thetherapeutic agent to an isolated polypeptide having from about 50 to 79amino acids taken from the sequence of SEQ ID NO. 1, wherein thepolypeptide binds to the extracellular domain ECD of HER-2 at anaffinity of at least 10⁸. Preferably, the isolated polypeptide is fromabout 69 to 79 amino acids in length. Preferably, the isolatedpolypeptide binds to a site on the ECD of HER-2 that is different fromthe site of binding of HERCEPTIN® (a marketed humanized monoclonalantibody that is used for the treatment of cancer and that binds to theECD or HER-2). It was discovered that the 79 amino acid polypeptide [SEQID NO. 1] exhibited surprising high affinity binding properties to theECD of HER-2. Moreover, the site of such binding is different andunaffected by the site of binding of a marketed humanized monoclonalantibody (HERCEPTIN®). Therefore, the high binding affinity enables the79 amino acid polypeptide to function as a targeting molecule to tumorcells expressing HER-2.

Anti-p68 Antibody as a Diagnostic/Prognostic Agent

The p68HER-2 ECDIIIa variant 3 (see TABLE 1, below) glycosylatedpolypeptide was expressed and used as an antigen for antibodyproduction. Specifically, antibody specific for p68HER-2 was prepared byinjecting rabbits with purified polyhistidine-tagged ECDIIIa variant 3peptide, which is the same as the intron encoded novel C-terminus orp68HER-2, the domain that binds with high affinity to p185HER-2. Theisolated polyclonal antibody detected pM quantities of ECDIIIa peptideor of p68HER-2 with high specificity (see FIGS. 3 and 5). Thus, anantibody specific for p68HER-2 is useful as a diagnostic agent fordetecting p68HER-2 in bodily fluids and tumor tissues using diagnostictechniques, such as ELISA, immunoprecipitations, immunohistochemistry orWestern analysis.

Antibodies that specifically recognize one or more epitopes of ECDIIIa,or epitopes of p68HER-2, or peptide fragments, and thus distinguishamong ECDIIIa variants (see TABLE 1, below) are also encompassed by theinvention. Such antibodies include but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single-chain antibodies, Fab fragments, F(ab′).sub.2fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. The antibodies of the invention may be used, forexample, in the detection of a particular p68HER-2 ECDIIIa variant in abiological sample and may, therefore, be utilized as part of adiagnostic or prognostic technique whereby patients or tissue samplesmay be tested for the presence of particular variants, or for abnormalamounts particular variants.

Such antibodies may also be utilized in conjunction with, for example,compound screening schemes for the evaluation of the effect of testcompounds on expression and/or activity of particular p69HER-2 variants.Additionally, such antibodies can be used in conjunction with the cancertreatment methods described herein.

For the production of antibodies, various host animals may be immunizedby injection with e.g., polyhistidine-tagged ECDIIIa variantpolypeptides, truncated ECDIIIa variant polypeptides, functionalequivalents of the ECDIIIa variants or mutants of the ECDIIIa region.Such host animals may include but are not limited to rabbits, mice,hamsters and rats, to name but a few. Various adjuvants may be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of the immunized animals. Monoclonalantibodies, which are homogeneous populations of antibodies to aparticular antigen, may be obtained by any technique that provides forthe production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (Nature 256:495-497, 1975; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al.,Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA80:2026-2030, 1983), and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985). Such antibodies may be of any immunoglobulin class including IgG,IgM, IgE, IgA, IgD and any subclass thereof. Hybridomas producing mAbmay be cultivated in vitro or in vivo. Production of high titers of mAbsin vivo makes this the presently preferred method of production.

Additionally, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855,1984; Neuberger et al., Nature, 312:604-608, 1984; Takeda et al.,Nature, 314: 452-454, 1985) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion (humanized).

Alternatively, techniques described for the production of single-chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426, 1988;Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Wardet al., Nature 334:544-546, 1989) can be adapted to produce single-chainantibodies against ECDIIIa variant gene products. Single-chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′).sub.2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′).sub.2fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., Science, 246:1275-1281, 1989) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

Antibodies to particular ECDIIIa variants can, in turn, be utilized togenerate anti-idiotype antibodies that “mimic” the ECDIIIa variant,using techniques well known to those skilled in the art. (Greenspan &Bona, FASEB J 7 (5):437-444, 1993; and Nissinoff, J. Immunol.147:2429-2438, 1991). For example antibodies which bind to an ECDIIIavariant and competitively inhibit the binding of p68HER-2 to HER-2receptor can be used to generate anti-idiotypes that “mimic” the ECDIIIavariant and, therefore, bind and neutralize HER-2 receptor. Suchneutralizing anti-idiotypes or Fab fragments of such anti-idiotypes canbe used in cancer therapeutic regimens.

Alternatively, antibodies to particular ECDIIIa variants that can act asagonists or antagonists of the ECDIIIa variant activity can begenerated. Such antibodies will bind to the ECDIIIa variant and modulatethe activity of p68HER-2 vis-à-vis p185HER-2 receptor-mediated signaltransduction. Such antibodies may be particularly useful for treatingparticular cancers and/or modulating tumor differentiation. Accordingly,the present invention further provides a method for determining theprognosis of tumor treatment for a tumor that overexpresses HER-2,comprising: (a) obtaining a bodily fluid, wherein the bodily fluid isselected from the group consisting of blood, serum, urine, lymph,saliva, tumor tissue, and combinations thereof; and (b) measuring theamount of p68HER-2 expressed using an anti-p68HER-2 antibody-basedassay, wherein the assay is selected from the group consisting of ELISA,immunoprecipitation, immunohistocytochemistry, and Western analysis.Preferably, the method for determining the prognosis of tumor treatmentfurther comprises measuring the amount of p185HER-2 ECD in the bodilyfluid, and determining a ratio between the amount of p68HER-2 andp185HER-2. The higher the ratio of p68HER-2:p185HER-2, the better thetreatment prognosis.

ECDIIIa region Variants as Diagnostic/Prognostic Agents

Example 11 (below) shows that the human sequence of intron 8 is bothproline-rich and polymorphic. Sequencing of genomic DNA from fifteendifferent individuals resulted in the identification of 10 variablesequence regions within Her-2 Intron 8. See SEQ ID NO:10; FIG. 8, andTable 1. FIG. 8 shows the most common nucleotide and correspondingpolypeptide sequences of intron 8. This region contains 10 differentpolymorphisms (marked by the letters W (2×), Y (3×), R, N, M, and S (2×)in SEQ ID NO:10; or marked by an “X” in FIG. 8) that result innonconservative amino acid substitutions (see legend to TABLE 1). Forexample, the polymorphism (G→C) at nucleotide position 161 (FIG. 8;TABLE 1) would result in a substitution of Arginine (R) for Proline (P)at amino acid residue #54 of SEQ ID NO:1, or residue #394 of SEQ IDNO:2. The N-terminal Glycine (G), designated as position 1 in FIG. 8 orSEQ ID NO:10, corresponds to amino acid residue 341 in the “herstatin”sequence (Doherty et al., Proc. Natl. Acad. Sci. USA 96:10,869-10,874,1999). The nucleotide sequence shown in FIG. 1(A) (Doherty et al., Proc.Natl. Acad. Sci. USA 96:10,869-10,874, 1999), is a polymorphic form thatdiffers at amino acid residues #6 and #73 from the most commonlydetected sequence shown here in FIG. 8.

This result demonstrates that in the human population there are severalvariations in the intron-8 encoded domain that could lead to alteredbiochemical and biological properties among ECDIIIa-containing proteinvariants. An individual may, inter alia, be genetically heterozygous fortwo variants, homozygous for a given variant, or homozygous for a doublevariant. Both tumor progression and optimal treatment may vary dependingupon the particular variants represented in a given individual.

This variability has both prognostic and diagnostic utility. The presentinvention shows that ECDIIIa-containing polypeptides can bind tightlyto, and thus antagonize the HER-2 receptor. Such a specific,high-affinity interaction is dependent upon particular primary,secondary and tertiary structure of the ECDIIIa-containing polypeptide.The ECDIIIa region is proline-rich, and it is well known in the art thatnonconservative substitution of proline residues, or other residueswithin a proline-rich sequence, in a given protein can have profoundeffects on its secondary and tertiary structure. Thus, the polymorphismsof the present invention are likely to embody significant structural,biochemical and biological differences relative to the most commonpolypeptide structure (shown in FIG. 8). Structural differences amongECDIIIa variant proteins may include for example, differences in size,electronegativity, or antigenicity. Differences in biological propertiesamong ECDIIIa variants might be seen e.g., in the relative degree ofcellular secretion, the nature and/or extent of modulation of the HER-2receptor, pharmacokinetics (e.g., serum half-life, elimination profile),resistance to proteolysis, N-linked glycosylation patterns, etc. Thesebiological differences, in turn, would be expected to alter tumorprogression and thus optimal treatment protocols. Thus, the knowledgethat an individual contains a particular ECDIIIa variant or variants(e.g., in individuals heterozygous for a given variant, or individualswith compound variants like variant 11 of Table 1), may, in itself, beprognostic of particular cancer susceptibility.

The apparent genetic heterogeneity of ECDIIIa region means that thenature of the particular ECDIIIa variation carried by an individual mayhave to be ascertained using sequence identity assays prior toattempting genetic diagnosis of the patient. The analysis can be carriedout on any genomic DNA derived from bodily fluids of the patient,typically a blood sample from an adult or child, but alternatively maybe serum, urine, lymph, saliva, tumor tissue, placental tissue,umbilical cord tissue, amniotic fluid, and chorionic villi samples. Itis expected that standard genetic diagnostic methods, such ashybridization or amplification assays, can be used. Either DNA or RNA,may, for example, be used in hybridization or amplification assays ofbiological samples to detect particular ECDIIIa variant sequences. Suchsequence identity assays may include, but are not limited to, Southernor Northern analyses, single-stranded conformational polymorphismanalysis, in situ hybridization assays, and polymerase chain reaction(“PCR”) analyses. Such analyses may reveal both quantitative andqualitative aspects of ECDIIIa variant sequence expression. Such aspectsmay include, for example, point mutations, and/or activation orinactivation of gene expression. Standard in situ hybridizationtechniques may be used to provide information regarding which cellswithin a given tissue express a particular ECDIIIa variant sequence.

Preferably, diagnostic methods for the detection of ECDIIIa variantnucleic acid molecules involve contacting and incubating nucleic acids,derived from cell types or tissues being analyzed, with one or morelabeled nucleic acid reagents, or probes, specific for particularECDIIIa variants. More preferably, PCR, or reverse transcription PCR,can be utilized to identify nucleotide variation within the ECDIIIadomain. PCR reaction conditions should be chosen which optimizeamplified product yield and specificity, and, additionally, produceamplified products of lengths that may be resolved utilizing standardgel electrophoresis techniques. Such reaction conditions are well knownto those of skill in the art, and important reaction parameters include,for example, length and nucleotide sequence of oligonucleotide primers,and annealing and elongation step temperatures and reaction times.Following the PCR reaction, the PCR products can be analyzed by methodssuch as heteroduplex detection, cleavage of RNA-DNA hybrids using RnaseA, single-stranded conformational polymorphisms, and denaturing gradientgel electrophoresis.

Additionally, if the particular ECDIIIA sequence variant is known to addor remove a restriction site, or to have significantly altered the sizeof a particular restriction fragment, a protocol based upon restrictionfragment length polymorphism (“RFLP”) analysis may be appropriate.

ECDIIIa variants can also be analyzed at the expression level usingsequence identity assays with bodily fluids derived from the patient,typically a blood sample from an adult or child, but may include serum,urine, lymph, saliva, tumor tissue, placental or umbilical cord cells,amniotic fluid, and chorionic villi samples. Well-known sequenceidentity assays for analyzing expression include, but are not limitedto, mRNA-based methods, such as Northern blots and in situ hybridization(using a nucleic acid probe derived from the relevant cDNA), andquantitative PCR (as described by St-Jacques et al., Endocrinology134:2645-2657, 1994).

Polypeptide-based methods (e.g., including but not limited to westernblot analysis) including the use of antibodies specific for the ECDIIIavariant of interest, as discussed above, could also be used. Thesetechniques permit quantitation of the amount of expression of a givenECDIIIa variant, at least relative to positive and negative controls.Preferably, a battery of monoclonal antibodies, specific for differentECDIIIa eptitopes or variants, could be used for rapidly screening cellsor tissue samples to detect those expressing particular ECDIIIavariants, or for quantifying the level of ECDIIIa variant polypeptides.Preferred diagnostic methods for the quantitative or qualitativedetection of ECDIIIa variant peptide molecules may involve, for example,immunoassays wherein particular ECDIIIa-containing peptides are detectedby their interaction with anti-ECDIIIa variant specific antibodies. Thiscan be accomplished for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody coupled with lightmicroscopic, flow cytometric, or fluorometric detection. The antibodies(or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of ECDIIIa-containingpeptides. Through the use of such procedures, it is possible todetermine not only the presence of particular ECDIIIa-containingpolypeptides, but also their distribution in the examined tissue.

Immunoassays for ECDIIIa variant polypeptides preferably compriseincubating a biological sample, such as the above-named bodily fluids,which have been incubated in the presence of a detectably labeledantibody capable of identifying ECDIIIa-containing peptides, anddetecting bound antibody by any of a number of techniques well known inthe art. The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support that is capable of immobilizingsoluble proteins, cells, or cell particles. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled anti-ECDIIIa variant specific antibody. The solid phase supportmay then be washed with the buffer a second time to remove unboundantibody. The amount of bound label on the solid support may then bedetected by conventional means.

Alternatively, anti-ECDIIIa variant specific antibodies can bedetectably labeled by linking the same to an enzyme for use in an enzymeimmunoassay or Enzyme Linked Immunosorbent Assay (“ELISA”). The enzymewhich is bound to the antibody will react with an appropriate substrate,preferably, a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetirc or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.

The detection can be accomplished by calorimetric methods that employ achromogenic substrate for the enzyme. Detection may also be accomplishedvisually by comparison of the extent of enzymatic reaction withappropriate standards. Detection may also be accomplished using any of avariety of other immunoassays. For example, by radioactively labelingthe antibodies or antibody fragments, it is possible to detectECDIIIa-containing peptides through the use of a radioimmunoassay (RIA).The radioactive isotope can be detected by such means as the use of agamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can be detected due to fluorescence.Among the most commonly used fluorescent labeling compounds arefluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylene diaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound may be used to labelthe antibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

The binding activity of a given lot of anti-ECDIIIa-variant specificantibody may be determined according to well-known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Accordingly, the present invention, including the unexpected discoveryof a plurality of variable sequence positions within the proline-richECDIIIa region, along with antibodies specific for particular ECDIIIavariants, provides for valuable prognostic and diagnostic informationand assays.

Accordingly, the present invention further provides a method fordetermining the prognosis of tumor treatment in a patient for a tumorthat overexpresses HER-2, comprising: (a) obtaining a bodily fluidsample from the patient, wherein the bodily fluid is selected from thegroup consisting of blood, serum, urine, lymph, saliva, tumor tissue,placental tissue, umbilical cord tissue, amniotic fluid, chorionic villitissue, and combinations thereof; and (b) measuring the amount ofp68HER-2 expressed using an anti-p68HER-2 antibody-based assay, whereinthe assay is selected from the group consisting of ELISA,immunoprecipitation, immunohistocytochemistry, and Western analysis.Preferably, the method for determining the prognosis of tumor treatmentfurther comprises measuring the amount of p185HER-2 ECD in the bodilyfluid, and determining a ratio between the amount of p68HER-2 andp185HER-2. The higher the ratio of p68HER-2:p185HER-2, the better thetreatment prognosis. Preferably, the method for determining theprognosis of tumor treatment further comprises determining whichparticular ECDIIIa variants are present and optimizing tumor treatmentin view of particular biochemical and biological properties amongECDIIIa protein variants.

The present invention further provides an assay for cancer treatment,prognosis or diagnosis in a patient comprising: (a) obtaining a bodilyfluid sample from the patient, wherein the bodily fluid is selected fromthe group consisting of blood, serum, urine, lymph, saliva, tumortissue, placental tissue, umbilical cord tissue, amniotic fluid,chorionic villi tissue and combinations thereof; (b) determining whethera particular ECDIIIa variant sequence is present in the bodily fluidsample with a sequence identity assay; and (c) correlating the presenceof the ECDIIIa variant sequence to cancer treatment and diagnosis usingan historical database. Preferably, the sequence identity assay isselected from the group consisting of DNA sequencing, PCR assays, ELISAimmunologic assays, immunoassays, hybridization assays, and combinationsthereof.

The present invention further provides an assay for cancer treatment,prognosis or diagnosis in a patient comprising: (a) obtaining a bodilyfluid sample from the patient, wherein the bodily fluid is selected fromthe group consisting of blood, serum, urine, lymph, saliva, tumortissue, placental tissue, umbilical cord tissue, amniotic fluid,chorionic villi tissue and combinations thereof; (b) determining whetheran amount of an p68HER-2 ECDIIIa variant is present in the bodily fluidsample using an anti-p68HER-2 antibody-based assay, wherein the assay isselected from the group consisting of ELISA, immunoprecipitation,immunohistocytochemistry, and Western analysis; and (c) correlating thepresence or amount of the p68HER-2 ECDIIIa variant to cancer treatmentand diagnosis using an historical database.

The present invention further provides for the above-mentioned cancertreatment, prognostic or diagnostic assays, further comprising measuringthe amount of p185HER-2 ECD in the bodily fluid sample.

The present invention further provides for the above-mentioned cancertreatment, prognostic or diagnostic assays further comprising measuringthe amount of p185HER-2 ECD in the bodily fluid sample, and determininga ratio between the amount of p185HER-2 ECD and a particular p68HER-2ECDIIIa variant.

The present invention further provides for antibodies specific forECDIIIa variants of the sequence in SEQ ID NO:1 or SEQ ID NO:2, below.

P68HER-2 as a Therapeutic Agent

Without being bound by theory, but it appears that p68HER-2 or ECDIIIapeptide inhibits the growth of tumor cells that overexpress HER-2 bybinding to p185HER-2 at the cells surface. This hypothesis was examinedby testing anchorage independent growth of cells in the presence orabsence of p68HER-2 using cells that depend on p185HER-2 overexpressionfor their malignant growth, yet have little or no detectable p68HER-2.Anchorage independent growth of cells in soft agar was used as apredictive model for tumor cytotoxicity. This is a common and predictiveprocedure to examine transforming activity and reflects the tumorigenicand oncogenic potential of cells (DiFore et al., Science 237:178-182,1987; Hudziak et al., Proc. Natl. Acad. Sci. USA 84:7159-7163, 1987; andBaasner et al., Oncogene 13:901-911, 1996).

The effects of p68HER-2 on anchorage independent growth in soft agar wasdetermined using SKOV-3 carcinoma cells and HER-2 transfected 17-3-1cells, which are both tumorigenic and overexpress p185HER-2. The cellswere suspended in media supplemented with fetal calf serum in thepresence or absence of p68HER-2 and incubated for 21 days in ahumidified incubator. Anchorage independent growth was quantitated bycounting the number of colonies that contained more than 50 cells. FIG.7 shows that in the presence of p68HER-2, anchorage independent growthof both SKOV-3 cells and 17-3-1 cells was inhibited several fold.Accordingly, these data show that p68HER-2 is not just cytostatic, butcytotoxic and possibly apoptotic.

Example 1

This example provides the results from an experiment to investigateHER-2 mRNA diversity within the extracellular domain (ECD) codingsequence using polymerase chain reaction (PCR). A cDNA library fromSKOV-3 cells (American Type Culture Collection (Rockville, Md.)maintained in DMEM, supplemented with 10% fetal bovine serum and 0.05%gentamycin), an ovarian carcinoma cell line in which the HER-2 gene isamplified eight times (Tyson et al., Am. J. Obstet. Gynecol.165:640-646, 1991) was examined using a forward primer specific for exon1 (Tal et al., Mol. Cell. Biol. 7, 2597-2601, 1987) identical tonucleotides 142-161 and a reverse primer complementary to nucleotides1265-1286 in exon 9 (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993).Briefly, The SKOV-3 cDNA library was provided by Origene Technologies,Inc. (Rockville, Md.), and was prepared from RNA extracted from SKOV-3cells. RNA was extracted from SKOV-3 cells grown to 80% confluence on 15cm plates with TriReagent (Molecular Research Center, Inc., Cincinnati,Ohio), according to the manufacturer's protocol, to obtain total RNA.RNA was resuspended in 10 mM Tris-EDTA, pH 8.0, for reversetranscription and cDNA library construction, or in RNA hybridizationbuffer (80% formamide, 40 mM PIPES, 4 mM NaCl, 1 mM EDTA, pH 7.5) forribonuclease protection assay (RPA). RNA concentrations were determinedspectrophotometrically at OD₂₆₀. Poly A⁺ mRNA was selected from totalRNA using a mRNA extraction kit (Oligotex, Qiagen).

A product of ˜1420 bp, determined to be HER-2-specific by Southernblotting, was approximately 270 bp larger than the expected size of 1144bp from the previously described cDNA sequence (Coussens et al., Science230:1132-1139, 1985). Briefly, the Southern blotting proceduretransferred nucleic acids from agarose gels under vacuum (Bio-Rad Model785 Vacuum Blotter) in 0.4 M NaOH to Gene Screen Plus HybridizationTransfer Membrane (NEN Research Products, Boston, Mass.). Nucleic acidswere fixed to membranes by UV crosslinking in a UV-Stratalinker(Stratagene, Inc., La Jolla, Calif.), and the membranes were blocked inhybridization buffer (50% formamide, 5×SSC, 1% SDS, 10 mg/ml herringsperm DNA) at 42° C. for 2 h. The membranes were hybridized at 42° C.for 16 h in hybridization buffer with 10⁷ cpm of a 220 bp Kpn-HincIIfragment from ECDIIIa cDNA labelled with (α-³²P)dCTP (NEN Life Sciences)using a Random Prime DNA Labelling Kit (Boehringer Mannheim).

Templates were amplified in a Perkin Elmer GeneAmp PCR System 2400(Perkin Elmer Cetus, Emeryville, Calif.) using the Expand High FidelityPCR System (Boerhinger Mannheim) with 1× High Fidelity PCR buffercontaining 2.5 mM MgCl₂, 5 μM of each primer, and 200 μM dNTPs. Allprimers were obtained from GIBCO BRL (Life Technologies). Numbering ofnucleotide and amino acid residues is according to the HER-2 cDNAsequence reported by Coussens et al. (Coussens et al., Science230:1132-1139, 1985). The HER-2 extracellular domain was targeted foramplification from an SKOV-3 cDNA library (Origene Technologies, Inc.)using a forward primer (A) identical to nucleotides (nt) 142-161 ofHER-2 cDNA (5′-TGAGCACCATGGAGCTGGC-3′ [SEQ ID NO 3]), which spans theinitiation codon (underlined) and a reverse primer (B)(5′-TCCGGCAGAAATGCCAGGCTCC-3′ [SEQ ID NO 4]), which is complementary toHER-2 exon sequence at nt 1265-1286. Cycling parameters were: 94° C., 30sec; 58° C., 45 sec; 68° C., 3 min, for 30 cycles. The region spanningthe alternative sequence (denoted ECDIIIa) from genomic DNA, wasamplified using a forward primer (C) (5′-AACACAGCGGTGTGAGAAGTGC-3′ [SEQID NO 5]) identical to HER-2 exon-specific sequence at nt 1131-1152 andthe reverse primer (B) [SEQ ID NO. 4] on DNA prepared as described (Bondet al., FEBS Letters 367:61-66, 1995) with cycling parameters: 94° C.,30 sec; 62° C., 30 sec; 72° C., 60 sec, for 25 cycles.

Reverse transcriptase-polymerase chain reaction (RT-PCR) was used toinvestigate the structure of mRNA containing the ECDIIIa sequence. Firststrand cDNA was reverse transcribed (Bond et al., FEBS Letters367:61-66, 1995) using 5 μg RNA primed with 0.5 μg oligo-dT. To amplifythe ECDIIIa insert and adjacent 5′ HER-2 exon sequence, a forward primer(A) described above and a reverse primer (D)(5′-ATACCGGGACAGGTCAACAGC-3′[SEQ ID NO 6]) which is complementary to the3′ECDIIIa-specific sequence were used. Cycling parameters were: 94° C.,30 sec; 60° C., 40 sec; 68° C., 2 min, for 30 cycles.

Amplification of the ECDIIIa insert and adjacent 3′ HER-2 exon-specificsequence was with a forward primer (E) (5′-TCTGGGTACCCACTCACTGC-3′ [SEQID NO 7]) which is identical to the 5′ECDIIIa-specific sequence andcontains a Kpn1 restriction site and a reverse primer (F)(5′-TTCACACTGGCACGTCCAGACC-3′ [SEQ ID NO 8]) which is complementary toHER-2 exon sequence at nt 3898-3919 and spans the termination codon(underlined). Cycling parameters were: 94° C., 30 sec; 60° C., 40 sec;68° C., 5 min, for 30 cycles.

The PCR product was subcloned and the nucleotide sequence wasdetermined.

The results showed that the normal HER-2 coding sequence was presentbeginning with the 5′ primer sequence and continued uninterruptedthrough nucleotide 1171. At this position, a 274 nucleotide insertionwas found, followed by the expected coding sequence, including the 3′primer sequence. Analysis of the predicted protein product showed thatthe 274 nucleotide insertion encodes an extension of the known HER-2protein, beginning at residue 340 (Coussens et al., Science230:1132-1139, 1985), and introduces an in-frame stop codon 79 aminoacids later (FIG. 1). Comparison of the inserted nucleotides and theirpredicted amino acid sequence with sequences in Genbank showed nohomologies. Examination of the 5′ and 3′ junctions of the divergentsequence revealed consensus splice donor and acceptor sites (Sharp, andBurge, Cell 91:875-879, 1997) and include a pyrimidine tract andpotential branchpoint adenine residues near the 3′end of the insertsequence (FIG. 1). Thus, the inserted sequence is likely to be anintron.

Inspection of the predicted amino acid sequence of the novel 79 aminoacids [SEQ ID NO. 1] encoded by the inserted sequence shows a consensusN-linked glycosylation site and a high proline content of 19% (FIG. 1).The inserted sequence was designated ECDIIIa since it is located at theboundary between subdomains II and III in the extracellular domain ofthe p185HER-2 sequence (Lax et al., Mol. Cell. Biol. 8:1831-1834, 1988).The insert sequence is in-frame with the adjacent 5′ HER-2 exon sequencefor 236 nt where there is a termination codon.

Example 2

This example provides the results from experiments characterizingECDIIIa as contiguous with HER-2 exons in the genome. To investigate theHER-2 gene structure in the region of the ECDIIIa sequence, a forwardprimer, identical to nucleotides 763-785, and a reverse primer,complementary to nucleotides 1265-1286 of the HER-2 cDNA, were used inthe PCR on human genomic DNA. The amplification product was anticipatedto span exon 5 (Tal et al., Mol. Cell. Biol. 7:2597-2601, 1987) to anexon which is immediately 3′ of the ECDIIIa sequence. Intron number andsizes were estimated based on PCR product sizes, restriction digestanalysis, and partial sequence analysis of amplification products.

Next, human genomic DNA was examined using HER-2 exon-specific primersthat directly flank the insert to determine the sequences immediatelyflanking the ECDIIIa sequence. A ˜430 bp product was amplified fromnormal human genomic DNA and from genomic DNA extracted from carcinomacell lines SKOV-3, SKBR-3 and BT474, all of which have HER-2 geneamplification (Kraus et al., EMBO J. 6:605-610, 1987) and were found toexpress ECDIIIa in their cDNA. The identities of the PCR products asHER-2 were verified by Southern blot analysis using the proceduredescribed in Example 1. Nucleotide sequence analysis showed that the PCRproduct from human genomic DNA contained the ECDIIIa insert, flankedimmediately on both sides by known HER-2 coding sequence; no mutationsor rearrangements were seen. These data show that the ECDIIIa sequencerepresents a wholly retained intron, likely intron 8 based on the sizeof products amplified following intron 4 and on the location of intron 8in the homologous EGFR gene and HER-3 gene (Lee and Maihle, Oncogene16:3243-3252, 1998).

Example 3

This example shows that ECDIIIa is the only retained intron within thecoding sequence of HER-2 mRNA. To determine whether additional intronswere retained in the mRNA containing the ECDIIIa insert sequence, thereverse transcriptase-polymerase chain reaction (RT-PCR) was employed.First, a forward primer identical to 5′ HER-2 cDNA sequence at 142-161which spans the initiation codon, and a reverse primer complementary tothe 3′ ECDIIIa sequence were employed with SKBR-3 and SKOV-3 cDNA. Aproduct of 1.3 kb was amplified, which is the size expected if theproduct contained no introns other than intron 8. Amplification of the3′HER-2 coding sequence was then performed using a forward primeridentical to 5′ECDIIIa sequence and a reverse primer complementary to3′HER-2 cDNA sequence at nucleotides 3898-3919, which spans thep185HER-2 termination codon. A product of 2.9 kb was amplified, which isthe size expected from the HER-2 cDNA if no additional introns wereretained.

Further characterizations of both the 5′(1.3 kb) and 3′(2.9 kb)amplification products by restriction digest analysis and nucleotidesequencing confirmed the absence of additional retained introns. Todetermine the size of the products amplified when intron sequences areincluded, genomic DNA was used as a template for the PCR reactions,which resulted in products of approximately 10 kb for the 5′ codingsequence and 5 kb for the 3′ coding sequence. These results indicatethat the alternative HER-2 transcript, resulting from retention of anintron of 274 bp, was expected to be about 4.8 kb in size, assuming thatthe 5′untranslated (5′UTR) and 3′untranslated (3′UTR) regions areidentical in size to the previously described ˜4.5 kb HER-2 cDNA(Coussens et al., Science 230:1132-1139, 1985).

Example 4

This example illustrates the expression of a protein containing anECDIIIa sequence. To assess whether the alternative sequence istranslated into a protein product, the ECDIIIa sequence was expressed asa polyhistidine-tagged peptide in bacteria, purified the peptide bynickel-affinity chromatography, and raised antisera against the purifiedpeptide. Briefly, the bacterial expression vector was prepared byamplifying the ECDIIIa sequence from the SKOV-3 cDNA library usingprimer E and a reverse primer complementary to the 3′ end of the ECDIIIainsert sequence. The reverse primer contained a BamH1 restriction sitesequence, and was identical to that used for template construction inthe RPA (described in examples 1 and 2). The PCR amplification productof ˜280 bp was digested with Kpn1 and BamH1, gel purified (Qiaex II,Qiagen, Chatsworth, Calif.), and cloned into the pET30a vector, whichencodes a six histidine tag at the amino-terminus of the expressedprotein (Novagen, Madison, Wis.). The resulting expression vector,pET-ECDIIIa, was used for transformation of bacterial strain BL21.

To express the ECDIIIa protein product, BL21 cells transformed with thepET-ECDIIIa expression vector were grown in LB broth with 30 μg/mlKanamycin for 4 h at 37° C. Expression was induced with 0.1 mM IPTG for3 h and the harvested cells were lysed by sonication, and thencentrifuged at 39,000×g for 20 min. The supernatant was absorbed ontoNi-NTA agarose (Qiagen), by shaking for 60 min at room temperature. Theresin was washed with ten volumes of wash buffer (10 mM Tris pH 7.9 and300 mM NaCl), followed by ten volumes of wash buffer with 50 mMimidazole. The his-tagged ECDIIIa protein was eluted in wash buffer with250 mM imidazole. The his-tagged protein, which was estimated to beapproximately 90% pure by Coomassie Blue staining of gels, was used togenerate and characterize antibodies.

Briefly, anti-ECDIIIa antisera were produced by Cocalico Biologicals,Inc. (Reamstown, Pa.) by injection of two rabbits with purifiedpolyhistidine-tagged ECDIIIa peptide (described below). Polyclonalanti-neu (N) was produced against a peptide identical to amino acidresidues 151-165 of p185HER-2 (Lin and Clinton, Oncogene 6:639-643,1991). Polyclonal anti-neu (C) was made against a peptide identical tothe last 15 residues of the carboxy-terminus of p185HER-2 (Lin et al.,Mol. Cell. Endocrin. 69:111-119, 1990). Antisera from two immunizedrabbits were characterized and found to contain antibodies of high titerthat reacted with the purified ECDIIIa peptide.

A Western blot analysis examined whether SKBR-3 cells, which expressedthe alternative sequence in its cDNA, produced a protein that reactswith anti-ECDIIIa antibody. A 68 kDa protein from the cell extract andfrom the extracellular media reacted with anti-ECDIIIa antibody from twodifferent rabbits diluted at least 20,000 fold, but not with preimmunesera. Inspection of the cDNA sequence of the alternative transcript(FIG. 1) predicted a secreted protein product of 65-70 kDa if all 5consensus N-linked glycosylation sites in the N-terminal p185HER-2sequence were glycosylated (Stern et al., Mol. Cell. Biol. 6:1729-1740,1986).

If the 68 kDa ECDIIIa protein [SEQ ID NO. 2] is the translation productof the alternative HER-2 mRNA, then its N-terminal residues should beidentical to the N-terminal 340 residues of p185HER-2. Therefore, cellextract from SKBR-3 cells was immunoprecipitated with anti-peptideantibody against an N-terminal sequence of HER-2, anti-neu (N) (Lin andClinton, Oncogene 6:639-643, 1991) or with anti-ECDIIIa, and the immunecomplexes were examined by Western blot analysis with both antibodies.Briefly, three to 5 μl of antisera were added to 2 mg of protein fromcell lysates prepared in M-RIPA buffer (1% Nonidet P-40, 50 mM Tris pH7.4, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 1% aprotinin),which had been centrifuged to remove nuclei. Immunoprecipitation was for2 h with shaking at 4° C. as described (Lin et al., Mol. Cell. Endocrin.69:111-119, 1990). The immune complexes were bound to Protein GSepharose (Pharmacia) by incubation for 1 h at 4° C. with shaking,collected by centrifugation, and washed four times with M-RIPA. Theproteins were released from the immune complex by incubation at 95° C.for 2 min in SDS-PAGE sample buffer and resolved by SDS-PAGE in 7.5%gels (Mini-Protean II electrophoresis cell, Bio-Rad).

Western blotting was conducted following SDS-PAGE. Proteins wereelectroblotted onto nitrocellulose (Trans-blot, BioRad) using a semi-drytransfer unit (Bio-Rad) at 15 V for 20 min per gel (0.75 mm thick)equilibrated with 25 mM Tris pH 8.3, 192 mM glycine, 50 mM NaCl, and 20%methanol. The membranes were blocked with 5% nonfat dry milk at 25° C.for one hour. The blots were then incubated with primary antibody,washed twice for 15 min, and four times for 5 min with TBS-Tween(Tris-buffered saline containing 0.05% Tween), and then incubated for 40min with goat anti-rabbit secondary antibody, conjugated to horseradishperoxidase (Bio-Rad), diluted 1:10,000 in TBS-Tween. After incubationwith secondary antibody, the membranes were washed as described aboveand reacted with chemiluminescent reagent (Pierce) and then were exposedto Kodak X-OMAT BLU film.

As expected, p68HER-2 was detected when anti-ECDIIIa was used forimmunoprecipitation and for Western blot analysis. When anti-ECDIIIa wasused for immunoprecipitation and anti-neu (N) was the probe in theWestern blot, a 68 kDa protein was detected, indicating that p68ECDIIIacontained the N-terminal sequence of p185HER-2. Further, anti-neu (N)precipitated p68HER-2, which was detected by probing with anti-ECDIIIaantibody. These results demonstrate that p68HER-2 contains both ECDIIIaand the N-terminal sequence of HER-2.

Several other cell lines were examined for expression of p68ECDIIIa. Thecarcinoma cell lines which contained ECDIIIa sequence in their cDNA(BT474, SKOV-3) also had p68HER-2. Of several cell lines examined,HEK293 cells, derived from normal human embryonic kidney cells,expressed the highest levels of p68ECDIIIa in the cell extract and inthe extracellular media, at about 5 to 10-fold higher amounts thanSKBR-3 cells. In comparison to the carcinoma cell lines examined(SKBR-3, SKOV-3, and BT474) which overexpress p185HER-2, the HEK293cells contained about 20 fold lower amounts of p185HER-2. Therefore, therelative proportion of p68HER-2 to p185HER-2 was at least 100 foldgreater in HEK293 cells than in the three carcinoma cell lines studied.Reactivity with p68HER-2 as well as with a protein of ˜120 kDa,particularly apparent in the HEK293 extracts, was blocked bypreincubation of the antisera with purified ECDIIIa peptidedemonstrating sequence-specific reactivity. The larger protein may be adimer of p68HER-2. Therefore, p68HER-2 was expressed and secreted fromseveral carcinoma cell lines and is at 5-10 fold elevated levels inHEK293.

Example 5

This example illustrates expression of an alternative HER-2 transcriptcontaining the ECDIIIa intron sequence. Results of the RT-PCR analysisindicated that the ECDIIIa sequence was inserted into an otherwisenormal-sized HER-2 mRNA. These data suggest an alternative transcript of˜4.8 kb. To examine the size and expression of the ECDIIIa alternativetranscript, Northern blot analysis was conducted using anECDIIIa-specific probe. Briefly, a template for antisense RNA probesynthesis was constructed from SKOV-3 cDNA by PCR amplification of a 389bp sequence spanning the entire ECDIIIa insert sequence and containingadjacent 5′HER-2 exon sequence. The PCR was done using the forwardprimer C [SEQ ID NO. 5] that is identical to HER-2 cDNA sequence at nt1131-1152 and a reverse primer (5′-GCACGGATCCATAGCAGACTGAG GAGG-3′ [SEQID NO. 9]) which contains a 3′ BamH1 restriction endonuclease site andis complementary to the sequence spanning the 3′ splice site of theECDIIIa sequence. The PCR product was then digested with BamH1,liberating a 375 bp fragment, which was cloned into pBluescript SK(Stratagene). The plasmid was sequenced by the Vollum Institute CoreSequencing Facility (Portland, Oreg.) with m13 forward and reverseprimers. An antisense RNA probe complimentary to the entire ECDIIIasequence and to 87 nt of HER-2 exon sequence 5′ to the insert wastranscribed from 1 μg of linearized template using (α-³²P) CTP, T7 RNApolymerase, and the T7/SP6 Riboprobe Synthesis System (Promega, Madison,Wis.). This probe was expected to protect a 370 nt fragment whenhybridized with mRNA containing ECDIIIa and adjacent HER-2 exonsequence, and to protect an 87 nt fragment when hybridized with fullyspliced HER-2 mRNA.

To prepare the RNA hybrids, 30 μg of RNA were hybridized withapproximately 50,000 cpm of antisense RNA probe at 48° C. for 16 h. RNAhybrids were digested for 30 min at 37° C. with 40 μg/ml RNaseA(Boerhinger Mannheim) and 2 μg/ml RNase T1 (Life Technologies) in asolution of 250 mM NaCl, 5 mM EDTA, and 10 mM Tris pH 7.5. Proteinase K(100 μg) (Life Technologies) in 20 μl 10% SDS was added to stop thedigestion. Samples were extracted with acid phenol (pH 4.5; LifeTechnologies) and chloroform, precipitated with two volumes of 100%ethanol, and suspended in 5 μl of RPA sample buffer (88% formamide, 10mM EDTA pH 8.0, 1 mg/ml xylene cyanol, and 1 mg/ml bromophenol blue).Samples were denatured at 95° C. for 10 min and electrophoresed on a 5%polyacrylamide/urea gel in TBE (89 mM Tris, 89 mM borate, 2 mM EDTA pH8.3). Gels were dried under vacuum and subjected to phosphorimageranalysis for quantitation of the protected fragments (IP Lab Gel,Molecular Dynamics).

An alternative transcript of approximately 4.8 kb was detected in HEK293cells which expressed the highest levels of p68ECDIIIa. However analternative transcript could not be detected by Northern analysis of theSKBR-3, BT474, or SKOV-3 carcinoma cell lines. Therefore, the moresensitive ribonuclease protection assay (RPA) was employed to examinethe expression levels of the alternative transcript relative to thefully spliced 4.5 kb transcript. RNA from ovarian (SKOV-3) and breast(SKBR-3 and BT474) carcinoma cell lines, which contained detectablelevels of p68ECDIIIa, and a control cell line, 17-3-1, stablytransfected with HER-2 cDNA, were hybridized with an antisense³²P-labeled RNA probe which spanned the entire ECDIIIa (intron 8)sequence and 5′ HER-2 exon sequence flanking intron 8. Following RNasedigestion, electrophoresis, and autoradiography, a band of 370nucleotides was detected in each cell line except for 17-3-1, whichcorresponds to the expected size protected by an ECDIIIa-containingHER-2 mRNA. In addition, an 87 nucleotide protected fragment wasdetected in all cells and is the size expected for the fully-splicedHER-2 message which is overexpressed by more than 100 fold in thesecarcinoma cell lines compared to normal control cell lines (Kraus etal., EMBO J. 6:605-610, 1987). The amounts of each protected fragmentwere quantitated and normalized for size to estimate the relativeabundance of the alternative transcript, expressed as a percentage ofthe p185HER-2 mRNA. The alternative HER-2 mRNA with the ECDIIIa insertwas at 4.2% the level of the fully spliced transcript in SKOV-3; 5.4% inSKBR-3, and 0.8% in BT474 cells.

Example 6

This example shows that alternative transcripts containing the ECDIIIainsert were expressed in human embryonic kidney and liver. A Northernblot was conducted to examine whether an alternative transcript, whichcontains the ECDIIIa sequence, was expressed in normal human tissue.PolyA⁺ mRNA from a variety of human fetal tissues prepared as a Northernblot was hybridized with a radiolabeled probe specific for the uniqueECDIIIa sequence. A 4.8 kb mRNA was detected in kidney and a 2.6 kbtranscript was detected in liver (FIG. 2). The 4.8 kb transcript likelycorresponded to the full length 4.5 kb transcript with the 274 bp insertand the 2.6 kb transcript may have corresponded to a previouslydescribed 2.3 kb alternative transcript (Yamamoto et al., Nature319:230-234, 1986; and Scott et al., Mol. Cell. Biol. 13:2247-2257,1993) with the 274 bp ECDIIIa insert. When the blot was stripped andhybridized with a probe specific for the 5′ HER-2 coding sequence, abroad band representing the 4.8 and 4.5 kb mRNAs was detected in fetalkidney tissues and the truncated 2.6 kb transcript was detected in livershowing that these alternative transcripts contain sequences that encodethe HER-2 ECD. Because the inserted ECDIIIa sequence contained atermination codon, the same protein product may be produced from each ofthese mRNAs.

Several cell lines were also investigated for the ECDIIIa-containingalternative transcript by Northern blot analysis. The 4.8 kb alternativetranscript was detected in the human embryonic kidney cell line, HEK-293(FIG. 2). Although the ECDIIIa sequence was detected by RT-PCR analysisof SKBR-3, BT474, and SKOV-3 carcinoma cell lines, which all containHER-2 gene amplification, an ECDIIIa-containing alternative transcriptcould not be detected by Northern analysis of these cells. Therefore,the more sensitive ribonuclease protection assay (RPA) was employedusing an antisense probe which spanned the entire ECDIIIa sequence and5′ HER-2 exon sequence flanking the ECDIIIa sequence. The alternativeHER-2 mRNA with the ECDIIIa insert was detected at less than 5% of thefully spliced transcript in SKOV-3, SKBR-3, and BT474 cells. Thesefindings show that two alternative transcripts containing the ECDIIIasequence were expressed in a tissue-specific manner in normal humantissues, that the 4.8 kb alternative transcript was expressed in theHEK-293 cell line, and that the carcinoma cells with gene amplificationexpress reduced amounts of the alternative transcript at less than 5% ofthe 4.5 kb HER-2 transcript.

Example 7

This example illustrates expression of a protein containing the ECDIIIasequence. To assess whether the alternative sequence was translated intoa protein product, the ECDIIIa sequence, as a polyhistidine-taggedpeptide in bacteria, was expressed and purified by nickel-affinitychromatography, and raised antisera against the purified peptide. TheHEK-293 cells, which expressed the 4.8 kb ECDIIIa alternativetranscript, were examined for expression of an ECDIIIa-containingprotein by Western analysis. A 68 kDa protein from the cell extract andfrom the extracellular media reacted with the anti-ECDIIIa antibody(FIG. 3) but not with preimmune sera and reactivity was blocked bypreincubation of the antisera with purified ECDIIIa peptide (FIG. 3).The larger protein of ˜125 kDa detected in some cell extracts may be anaggregate of p68HER-2. The cDNA sequence of the alternative transcript(FIG. 1) predicts a secreted protein product of 65-70 kDa if all 5consensus N-linked glycosylation sites in the N-terminal p185HER-2sequence are glycosylated (Stern et al., Mol. Cell. Biol. 6:1729-1740,1986). Several other cell lines were examined for expression ofp68ECDIIIa. The carcinoma cell lines which contained ECDIIIa sequence intheir cDNA (BT474, SKOV-3, SKBR-3) also had detectable levels ofp68HER-2.

Example 8

This example illustrates the expression of p68HER-2 relative top185HER-2 was markedly reduced in carcinoma cell lines in which theHER-2 gene is amplified. Because the p68HER-2 mRNA was expressed at verylow levels relative to the p185HER-2 mRNA in carcinoma cell lines withHER-2 gene amplification, the relative proportions of p68HER-2 andp185HER-2 proteins in several cell lines were examined with and withoutHER-2 gene amplification. Western blots were prepared and probed withboth antisera specific for p68HER-2 and for p185HER-2. FIG. 4 shows thatp185HER-2 was readily detected in the carcinoma cells lines that havetheir HER-2 gene amplified about 8 times (Kraus et al., EMBO J.6:605-610, 1987). However, there was not a corresponding elevation inp68HER-2. In comparison, p68HER-2 was the only HER-2 protein detected inthe HEK-293, IOSEVAN, and HBL100 nontumorigenic cells, althoughp185HER-2 was expressed at very low levels in these cells (Kraus et al.,EMBO J. 6:605-610, 1987) and was detected in overexposed blots. Thesedata show that p68HER-2 was low in proportion to p185HER-2 in carcinomacells with HER-2 gene amplification and suggests that a mechanism mayexist to maintain low levels of p68HER-2 when p185HER-2 isoverexpressed.

Example 9

This example illustrates that p68HER-2 and the ECDIIIa peptidespecifically bind to p185HER-2. Because p68HER-2 is secreted andcontains subdomains I and II identical to p185HER-2, in addition to anovel sequence, the possibility that p68HER-2 may interact withp185HER-2 was investigated. Antipeptide antibody against the N-terminusof p185HER-2 and p68HER-2, anti-neu (N), or antibody specific forp185HER-2, anti-neu(C), were used for immunoprecipitations of SKBR-3carcinoma cells, which express low levels of p68HER-2 and overexpressp185HER-2. The immunoprecipitated material was prepared as a Westernblot and probed with both anti-ECDIIIa specific for p68HER-2 and withanti-neu(C). Anti-neu (N) immunoprecipitated both p68HER-2 and p185HER-2(FIG. 5A). In addition, antibodies specific for the C-terminus ofp185HER-2 immunoprecipitated p185HER-2 and coprecipitated p68HER-2 (FIG.5A), suggesting an interaction between the two proteins.

Since binding interactions between ECD sequences are very weak (Tzaharet al., EMBO J. 16:4938-4950, 1997; Fitzpatrick et al., FEBS Letters431:102-106, 1998), the possibility that binding may be conferred by thenovel proline rich ECDIIIa domain was examined. The unique 79 amino aciddomain, purified as a His-tagged protein, was immobilized on nickelagarose and used in a pull-down assay. For controls, two purifiedHis-tagged peptides unrelated to ECDIIIa, a 600 residue fragment of theWilson's disease membrane protein, and a 70 residue fragment containingthe DNA binding domain of the CREB protein, were likewise immobilized onnickel agarose resin. The immobilized peptides were incubated withprotein extracts prepared from HER-2 transfected 3T3 cells (17-3-1).Following extensive washes, the bound proteins were eluted and preparedas a Western blot which was probed with an antibody specific forp185HER-2. Equal amounts of His-tagged ECDIIIa peptide and controlpeptide were bound to the resin as confirmed by elution with 1Mimidazole and Coomassie staining of the eluted material in SDS-gels.While no p185HER-2 was retained by resin without peptide or with controlpeptide, p185HER-2 was selectively retained by the ECDIIIa peptide (FIG.5B).

Since the ECDIIIa domain bound to p185HER-2 in a pulldown assay, thequestion of whether the ECDIIIa domain preferentially binds to cellsthat overexpress p185HER-2 was examined. This was investigated usingmonolayer cultures of 17-3-1 cells transfected with HER-2 compared tothe parental 3T3 cells. The cells were incubated with differentconcentrations of the His-ECDIIIa peptide, washed, and extracted indenaturing buffer with protease inhibitors. To detect any bound peptide,the cell extracts were examined by Western blot analysis usingantibodies specific for ECDIIIa. In addition, equal aliquots of theECDIIIa peptide treated cells were reacted as a Western blot withantibodies specific for p185HER-2, demonstrating the overexpression ofp185HER-2 in the transfected 17-3-1 cells. The ECDIIIa peptidepreferentially bound to intact 17-3-1 cells at nM concentrations (FIG.5C) whereas little or no peptide was found to bind to equivalent amountsof parental 3T3 cells suggesting a specific interaction with theextracellular domain of p185HER-2.

Example 10

Effect of p68ECDIIIa and the ECDIIIa peptide on tyrosine phosphorylationof p185HER-2 was examined. Tyrosine phosphorylation of RTKs is theinitial indication of ligand activation and signal transduction.Tyrosine phosphorylation in 17-3-1 cells treated with different amountsof the purified ECDIIIa peptide, with conditioned media (CM) from HEK293cells that contained high levels of p68HER-2 (FIG. 2A), or with control,conditioned media from SKOV-3 cells that had no detectable p68HER-2 wereexamined. There was no increase in the tyrosine phosphorylation signalat 10 minutes (FIG. 6) or 2 hrs of treatment with His-ECDIIIa or withconcentrated CM suggesting that p185HER-2 was not activated. Neitherp68HER-2-containing CM nor the ECDIIIa peptide detectably altered thephosphotyrosine signal corresponding to p185HER-2 from SKOV-3 cells inwhich p185HER-2 tyrosine phosphorylation levels were low. Additionally,p68HER-2 and the ECDIIIa peptide had no discernable effect on in vitroself-phosphorylation activity of p185HER-2 immunoprecipitated from17-3-1 cell extracts. These results support the conclusion that p68HER-2did not activate p185HER-2 signal transduction.

Example 11

This example illustrates that the sequence of intron 8 is polymorphicwithin that portion of intron 8 that serves as in-frame (with theextracellular domain of p185HER-2) coding sequence when intron 8 isalternatively retained in mRNA.

Intron 8 of the human HER-2 gene is alternatively retained in mRNA, andencodes a novel 79-residue domain at the C-terminus of a part of theextracellular domain of p185HER-2. The product, “herstatin,” of thealternative transcript with the retained intron functions as anautoinhibitor of the HER-2 oncogene. The intron 8 encoded domain, alone,was shown to bind with nM affinity to p185HER-2. (Doherty et al., Proc.Natl. Acad. Sci. USA 96:10,869-10,874, 1999).

Polymorphisms in the nucleotide and deduced amino acid sequence ofintron 8 in the HER-2 gene were identified by sequencing genomic DNAfrom 15 different individuals. FIG. 8 and SEQ ID NO:1 show the mostcommon nucleotide and corresponding amino acid sequences, respectively,of intron 8. This region contains 10 different polymorphisms (marked bythe letters W (2×), Y (3×), R, N, M, and S (2×) in SEQ ID NO:10; ormarked by an “X” in FIG. 8) that result in nonconservative amino acidsubstitutions (see legend to TABLE 1). For example, the polymorphism(G→C) at nucleotide position 161 (FIG. 8; TABLE 1) would result in asubstitution of Arginine (R) for Proline (P) at amino acid residue #54of SEQ ID NO:1, or residue #394 of SEQ ID NO:2. The N-terminal Glycine(G), designated as position 1 in FIG. 8 or SEQ ID NO:10, corresponds toamino acid residue #341 in the “herstatin” sequence (Doherty et al.,Proc. Natl. Acad. Sci. USA 96:10,869-10,874, 1999). The nucleotidesequence shown in FIG. 1(A) is a polymorphic form that differs at aminoacid residues #6 and #73 from the most commonly detected sequence shownhere in FIG. 8.

This result demonstrates that in the human population there are severalvariations in the intron-8 encoded domain that could lead to alteredbiochemical and biological properties among ECDIIIa-containing proteinvariants. Some identified variants are summarized in Table 1:

TABLE 1 X(4) X(14) X(17) X(47) X(54) X(62) X(106) X(161) X(191) X(217)Variant 1 T Variant 2 C Variant 3 T Variant 4 A Variant 5 A Variant 6 C,T, A Variant 7 A Variant 8 G Variant 9 T Variant 10 C Variant 11 T CTable 1. Sequence variants in the intron-8 encoded domain found in thehuman population (based on 15 different individuals). Sequence variants1-11 are listed, showing the base changes at particular “X” positionsrelative to that found in the most common DNA sequence shown in FIG. 8.The numbers in parenthesis after each X correspond to the nucleotideposition in the DNA sequence shown in FIG. 8 or SEQ ID NO:10. The DNAsequence variants listed here correspond to the variable amino acidpositions (“Xaa”) of SEQ ID NO:1 as follows: X(4) to Xaa(2); X(14) toXaa(5); X(17) to Xaa(6); X(47) to Xaa(16); X(54) to Xaa(18); X(62) toXaa(21); X(106) to Xaa(36); X(161) to Xaa(54); X(191) to Xaa(64); X(217)to Xaa(73); and to the variable amino acid positions of SEQ ID NO:2 asfollows: X(4) to Xaa(342); X(14) to Xaa(345); X(17) to Xaa(346); X(47)to Xaa(356); X(54) to Xaa(358); X(62) to Xaa(361); X(106) to Xaa(376);X(161) to Xaa(394); X(191) to Xaa(404); X(217) to Xaa(413). The specificamino acid changes (relative to the most common DNA sequence of FIG. 8)for the variable amino acid positions in SEQ ID NO:1 are: Variant 1,Xaa(2)(Thr→Ser); Variant 2, Xaa(5) (Leu→Pro); Variant 3, Xaa(6)(Pro→Leu); Variant 4, Xaa(16) (Leu→Gln); Variant 5, Xaa(18) (Met→Leu);Variant 6, Xaa(21) (Gly→Asp, Alu or Val); Variant 7, Xaa(36) (Leu→Ile);Variant 8, Xaa(54) (Pro→Arg); Variant 9, Xaa(64) (Pro→Leu); Variant 10,Xaa(73) (Asp→Asn), and Variant 11, Xaa(6) (Pro→Leu) and Xaa(73)(Asp→Asn). The same substitutions apply to the corresponding variableamino acid positions in SEQ ID NO:2.

Example 12

This example shows (see Table III, below) five polymorphic HER-2 intron8 polymorphisms (sequence variants 12-16) identifiable in DNA samplesfrom African Americans.

Specifically, four polymorphic sites were identified within that portionof intron 8 that serves as in-frame (with the extracellular domain ofp185HER-2) coding sequence when intron 8 is alternatively retained inmRNA (i.e., four polymorphic sites within the sequence regionencompassed by SEQ ID NO:10, or within that encompassed by the sequenceregion of FIG. 8). Two of these polymorphic sites (variants 12 and 15)correspond in position to those (variants 3 and 10, respectively)disclosed above in Example 11, whereas the other two (variants 13 and14) represent additional polymorphic sites (Table II).

Furthermore, (see Table II and Table III, below) an additionalpolymorphic site (variant 16) was identified in a region of intron 8that remains as “non-coding” sequence when intron 8 is alternativelyretained in mRNA. This “non-coding” intron 8 polymorphic site is located3′, or downstream from that portion of intron 8 that contains the otherpolymorphic sites shown in this Example and Example 11, and that servesas in-frame (with the extracellular domain of p185HER-2) coding sequencewhen intron 8 is alternatively retained in mRNA.

Methods. Polymorphisms in the nucleotide and deduced amino acid sequenceof intron 8 in the HER-2 gene were identified by sequencing genomic DNA(using blood samples) from 215 individuals corresponding to 75 AfricanAmericans (Black), 135 Caucasians (White), one Asian American (Asian)and 4 Hispanics. As for Example 11, above, the N-terminal Glycine (G orGly) designated as position 1 in FIG. 8 or SEQ ID NO:1 or SEQ ID NO:10,corresponds to amino acid residue #341 in the “herstatin” sequence ofSEQ ID NO:2 or SEQ ID NO:13.

Results. Table II designates the nucleotide substitutions and the twoamino acid residue substitutions in the coding sequence of intron 8 anda third nucleotide substitution in a non coding sequence of intron 8using numbering corresponding to the entire “herstatin” protein sequence(SEQ ID NO:2 or SEQ ID NO:13):

TABLE II N Black 75 White 135 Asian 1 Hispanic/Latino 4 Total 215Herstatin Polymorphism Distributions Among Blacks Prostate CasesControls Other Cancers Arg357Cys (C1081T) wt (%) 24 (96) 32 (89) 13 (93)het (%) 1 (4) 2 (6) 1 (7) mut (%) 0 (0) 2 (6) 0 (0) total 25 36 14Arg371Ile (G1124T) wt (%) 24 (96) 36 (100) 14 (100) het (%) 1 (4) 0 (0)0 (0) mut (%) 0 (0) 0 (0) 0 (0) total 25 36 14 C1279T (3′UTR) wt (%) 24(96) 36 (100) 12 (93) het (%) 2 (8) 0 (0) 2 (7) mut (%) 0 (0) 0 (0) 0(0) total 25 36 14Table II. This table shows the distribution of three additional(relative to those identified in Example 11) polymorphic regions inHER-2 intron 8 of the DNA from African American individuals. Amino acidsposition designations correspond to amino acid positions in the“Herstatin” sequence (SEQ ID NO:2 or SEQ ID NO:13).

Table III, below, illustrates that the sequence data revealedpolymorphisms at nucleotide positions #17 and #217 (also correspondingto nucleotide positions of the sequence region shown in FIG. 8 or SEQ IDNO:10). The polymorphism at position #17 (variant 12) corresponds tovariant 3 of Table I (Example 11). The polymorphism at position #217(variant 15) corresponds (at least at the protein level) to variant 10of Table I (Example 11) (see SEQ ID NO:12 and SEQ ID NO:13).

Additionally, the sequence data (see SEQ ID NO:11) revealed (see TableIII) that intron 8 contains three polymorphic sites (corresponding tovariants 13, 14 and 16) in addition to those disclosed in Example 11,above. Two of these (variants 13 and 14) are located at nucleotidepositions #49 and #92 of SEQ ID NO:11 (also corresponding to nucleotidepositions #49 and #92 of SEQ ID NO:10 (or FIG. 8). The third (variant16) is located at a nucleotide position #259 of SEQ ID NO:11 [alsocorresponding to nucleotide position #259 relative to the sequenceregion of SEQ ID NO:10 (or to position #264 of the sequence shown inFIG. 1, panel A)]. Thus, the polymorphism corresponding to variant 16 islocated 19 nucleotide positions 3′ (downstream) from that portion ofintron 8 that contains the other polymorphic sites shown in this Exampleand Example 11 (i.e., that portion represented by SEQ ID NO:10), andthat serves as in-frame (with the extracellular domain of p185HER-2)“coding” sequence when intron 8 is alternatively retained in mRNA.

Two of these polymorphisms result in nonconservative amino acidsubstitutions (see Table II and Table III, and legend of Table III; alsosee SEQ ID NO:12 and SEQ ID NO:13). For example, the polymorphism (C→T)found at the nucleotide position corresponding to nucleotide #49 of SEQID NO:11 [or to position #49 of SEQ ID NO:10 or FIG. 8] (i.e., thepolymorphism at position X(49) of Table 2) would result in asubstitution of Arginine (Arg) for Cysteine (Cys) at the amino acidposition corresponding to amino acid residue #17 of SEQ ID NO:12, SEQ IDNO:1, SEQ ID NO:10 or SEQ ID NO:11) or to amino acid residue #357 of SEQID NO:13 or SEQ ID NO:2.

SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 show the four variant aminoacid positions described in this example, along with those of Example 11that are also shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:10.

Table III designates (in addition to variants 12 and 15, whichcorrespond to variants 3 and 10, respectively of Table I) the nucleotidesubstitutions and the corresponding two additional (relative to those ofTable I of Example 11) amino acid residue substitutions (i.e., variants13 and 14) in the “coding” sequence of intron 8, along with the thirdnucleotide substitution in the 3′ “non-coding” region of intron 8. Thenumbers in parenthesis after each X (polymorphic position) refer tonucleotide positions of SEQ ID NO:11 [or, as in Table I, correspond to(or are relative to, in the case of X(259) the nucleotide positions inthe DNA sequences shown in FIG. 8 or SEQ ID NO:10].

As for Example 11, above, the N-terminal Glycine (G or Gly) designatedas position 1 in SEQ ID NO:11, FIG. 8, SEQ ID NO:1 or SEQ ID NO:10,corresponds to amino acid residue #341 in the “herstatin” sequence ofSEQ ID NO:2.

TABLE III X(17) X(49) X(92) X(217) X(259) Variant 12 T Variant 13 TVariant 14 T Variant 15 A Variant 16 TTable III. Sequence variants in the intron-8 encoded domain found inhuman tissues (based on 215 different individuals). Sequence variants12-16 are listed. The numbers in parenthesis after each X (polymorphicposition) refer to nucleotide positions of SEQ ID NO:11 [or to positionsthat correspond to, or are relative to (in the case of X(259)) thenucleotide positions in the DNA sequences shown in FIG. 8 or SEQ IDNO:10]. The DNA sequence variants listed here and in SEQ ID NO:11correspond to variable amino acid positions shown in SEQ ID NO:12 [andalso correspond to variable amino acid positions (“Xaa”) of SEQ ID NO:1or SEQ ID NO:10 as follows: X(17) to Xaa(6); X(49) to Xaa(17); X(92) toXaa(31); X(217) to Xaa(73)]. The DNA sequence variant X(259) occurs inan untranslated region, and therefore does not alter the amino acidsequence of herstatin. Likewise, the variants of this table correspondto variable amino acid positions of SEQ ID NO:13 and SEQ ID NO:2 asfollows: X(17) to Xaa(346); X(49) to Xaa(357); X(92) to Xaa(371); X(217)to Xaa(413). The specific amino acid changes (relative to the mostcommon DNA sequence of FIG. 8) for the variable amino acid positions inSEQ ID NO:11 and SEQ ID NO:12 are: Variant 12, Xaa(6)(Pro→Leu); Variant13, Xaa(17) (Arg→Cys); Variant 14, Xaa(31) (Arg→Ile); Variant 15,Xaa(73) (Asp→Asn). Variant 16, X(259) is in an untranslated region anddoes not code for an amino acid alteration, but instead alters only thenucleotide sequence at nucleotide position 259 (i.e., C→T). The samesubstitutions apply to the corresponding variable amino acid positionsin SEQ ID NO:13.

SKOV3 ovarian carcinoma cells. Two additional polymorphisms were foundin a cell line derived from human ovarian cancer (SKOV3). These twopolymorphisms result in nonconservative amino acid substitutions. Onepolymorphism is a substitution (C-T) at nucleotide #17 in the intron 8sequence and nucleotide #1037 in the “herstatin” sequence resulting in asubstitution of leucine for proline at amino acid residue #6 in theintron 8 sequence and at amino acid residue #346 in the “herstatin’sequence (i.e., of SEQ ID NO:2 or SEQ ID NO:13). The second polymorphismfound in the SKOV3 ovarian carcinoma cells line is a substitution (G-A)at nucleotide #217 in the intron 8 sequence and nucleotide #1237 in the“herstatin” sequence resulting in a substitution of Asparagine forAspartic Acid at amino acid residue #73 in the intron 8 sequence andamino acid #413 in the “herstatin” sequence (i.e., of SEQ ID NO:2 or SEQID NO:13).

Significantly, the five polymorphic sites identified in the sequenceanalysis of this Example 12 were found only in DNA samples from AfricanAmericans (Black).

Summary of Examples 11 and 12

Together, Examples 11 and 12 of the present invention disclose 13polymorphic positions in intron 8 of the Her-2 gene. Example 12,involved a relatively large DNA sample size, and indicated that the fivepolymorphic sites identified (three of which are distinct from the tenpolymorphic sites identified in Example 11) are unique to AfricanAmericans (Black). Twelve of the thirteen polymorphisms (i.e., exceptfor variant 16 of Example 12) of these two Examples are present in thatportion of intron 8 that serves as in-frame (with the extracellulardomain of p185HER-2) coding sequence when intron 8 is alternativelyretained in mRNA.

The polymorphism corresponding to variant 16 is located in a region ofintron 8 that remains as “non-coding” sequence when intron 8 isalternatively retained in mRNA. This “non-coding” intron 8 polymorphicsite is located 19 nucleotide positions 3′, or downstream from thatportion of intron 8 that contains the other polymorphic sites, and thatserves as in-frame (with the extracellular domain of p185HER-2) codingsequence when intron 8 is alternatively retained in mRNA.

These HER-2 intron 8 polymorphisms provide for novel DNA and proteinsequences, novel pharmaceutical compositions for treating solid tumorsthat overexpress HER-2, and monoclonal antibodies that bind to ECDIIIavariants corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:12 or SEQID NO:13. These HER-2 intron 8 polymorphisms also provide for prognosticand diagnostic assays for the treatment and prevention of cancer.

1.-23. (canceled)
 24. A method for determining the prognosis of tumortreatment in a patient for a tumor that overexpresses HER-2, comprising:(a) obtaining a bodily fluid sample from a patient, wherein the bodilyfluid is selected from the group consisting blood, serum, urine, lymph,saliva, tumor tissue, placental tissue, umbilical cord tissue, amnioticfluid, chorionic villi tissue and combinations thereof; and (b)measuring the amount of p68HER-2 expressed using an anti-p68HER-2antibody-based assay, wherein the assay is selected from the groupconsisting of ELISA, immunoprecipitation, immunohistocytochemistry, andWestern analysis.
 25. The method for determining the prognosis of tumortreatment for a tumor that overexpresses HER-2 of claim 24, furthercomprising measuring the amount of p185HER-2 ECD in the bodily fluid.26. The method for determining the prognosis of tumor treatment for atumor that overexpresses HER-2 of claim 25, further comprisingdetermining a ratio between the amount of p68HER-2 and p185HER-2,whereby the higher the p68HER-2 to p185HER-2 ratio, the better theprognosis of the patient.
 27. An assay for cancer treatment, prognosisor diagnosis in a patient comprising: (a) obtaining a bodily fluidsample from the patient, wherein the bodily fluid is selected from thegroup consisting of blood, serum, urine, lymph, saliva, tumor tissue,placental tissue, umbilical cord tissue, amniotic fluid, chorionic villitissue and combinations thereof; (b) determining whether an ECDIIIavariant protein or DNA sequence, or a HER-2 intron 8 variant DNAsequence is present in the bodily fluid sample using a sequence identityassay; and (c) correlating the presence of the ECDIIIa variant proteinor DNA sequence, or the HER-2 intron 8 variant DNA sequence to cancertreatment and diagnosis using an historical database.
 28. The diagnosticassay of claim 27, wherein the sequence identity assay is selected fromthe group consisting of DNA sequencing, PCR assays, ELISA immunologicassays, immunoassays, hybridization assays, and combinations thereof.29. The diagnostic assay of claim 27, further comprising measuring theamount of p185HER-2 ECD in the bodily fluid.
 30. An assay for cancertreatment, prognosis or diagnosis in a patient comprising: (a) obtaininga bodily fluid sample from the patient, wherein the bodily fluid isselected from the group consisting of blood, serum, urine, lymph,saliva, tumor tissue, placental tissue, umbilical cord tissue, amnioticfluid, chorionic villi tissue and combinations thereof; (b) determiningwhether a HER-2 intron 8 variant DNA sequence is present in the bodilyfluid sample using a sequence identity assay; and (c) correlating thepresence of the HER-2 intron 8 variant DNA sequence to cancer treatmentand diagnosis using an historical database.
 31. The diagnostic assay ofclaim 30, wherein the sequence identity assay is selected from the groupconsisting of DNA sequencing, PCR assays, hybridization assays, andcombinations thereof.
 32. The diagnostic assay of claim 30, furthercomprising measuring the amount of p185HER-2 ECD in the bodily fluid.33. An assay for cancer treatment, prognosis or diagnosis in a patientcomprising: (a) obtaining a bodily fluid sample from the patient,wherein the bodily fluid is selected from the group consisting of blood,serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilicalcord tissue, amniotic fluid, chorionic villi tissue and combinationsthereof; (b) determining whether an amount of an p68HER-2 ECDIIIavariant is present in the bodily fluid sample using an anti-p68HER-2antibody-based assay, wherein the assay is selected from the groupconsisting of ELISA, immunoprecipitation, immunohistocytochemistry, andWestern analysis; and (c) correlating the presence or amount of thep68HER-2 ECDIIIa variant to cancer treatment and diagnosis using anhistorical database.
 34. The diagnostic assay of claim 33, wherein thesequence identity assay is selected from the group consisting of DNAsequencing, PCR assays, ELISA immunologic assays, hybridization assays,and combinations thereof.
 35. The diagnostic assay of claim 33, furthercomprising measuring the amount of p185HER-2 ECD in the bodily fluid.36. The diagnostic assay of claim 35, further comprising determining aratio between the amount of p68HER-2 and p185HER-2 ECD.
 37. An antibodyspecific for an ECDIIIa variant of the sequence in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:12 or SEQ ID NO:13.
 38. An antibody specific forp68HER-2 ECDIIIa variant
 3. 39. A diagnostic kit comprising: (a) amonoclonal antibody or antigen-binding fragment thereof thatspecifically binds to an ECDIIIa variant of the sequence in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:12 or SEQ ID NO:13; and (b) a detectable label,whereby the binding of the antibody in step (a) can be detected.
 40. Thediagnostic kit of claim 36, wherein the label is selected from the groupconsisting of enzymes, radiolabels, chromohphores, chemiluminescenttags, and fluorescers.